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Sample records for hnh vector cho

  1. MAR characteristic motifs mediate episomal vector in CHO cells.

    Science.gov (United States)

    Lin, Yan; Li, Zhaoxi; Wang, Tianyun; Wang, Xiaoyin; Wang, Li; Dong, Weihua; Jing, Changqin; Yang, Xianjun

    2015-04-01

    An ideal gene therapy vector should enable persistent transgene expression without limitations in safety and reproducibility. Recent researches' insight into the ability of chromosomal matrix attachment regions (MARs) to mediate episomal maintenance of genetic elements allowed the development of a circular episomal vector. Although a MAR-mediated engineered vector has been developed, little is known on which motifs of MAR confer this function during interaction with the host genome. Here, we report an artificially synthesized DNA fragment containing only characteristic motif sequences that served as an alternative to human beta-interferon matrix attachment region sequence. The potential of the vector to mediate gene transfer in CHO cells was investigated. The short synthetic MAR motifs were found to mediate episomal vector at a low copy number for many generations without integration into the host genome. Higher transgene expression was maintained for at least 4 months. In addition, MAR was maintained episomally and conferred sustained EGFP expression even in nonselective CHO cells. All the results demonstrated that MAR characteristic sequence-based vector can function as stable episomes in CHO cells, supporting long-term and effective transgene expression. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. A bovine papillomavirus-1 based vector restores the function of the low-density lipoprotein receptor in the receptor-deficient CHO-ldlA7 cell line

    Directory of Open Access Journals (Sweden)

    Ustav Mart

    2002-04-01

    Full Text Available Abstract Background The rationale of using bovine papillomavirus-1 (BPV-1 derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7. Results The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles. Conclusion Bovine papillomavirus type-1 (BPV-1-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1–5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.

  3. [Construction of pGL3-SM22-SCAP (D443N) eukaryotic expression vector and its expression in CHO cells].

    Science.gov (United States)

    Wang, Yuanyuan; Hu, Jieli; Cui, Jing; Huang, Ailong; Ruan, Xiongzhong; Chen, Yaxi

    2010-01-01

    The experiment was designed to investigate the function of SREBP cleavage-activating protein (SCAP) mutant (D443N) by constructing an eukaryotic expressive vector using a smooth muscle specific promoter SM22 (pGL3-SM22-SCAP(D443N)). SM22 promoter (pSM22) was amplified from genome DNA of mice by nested PCR, and then cloned into pMD-T vector. The SM22 promoter fragment released from the vector by Kpn I and Hind III digestion was sub-cloned into pGL3-control-Luc vector, to form pGL3-SM22-Luc. The activity of pSM22 in human vascular smooth muscle cells (VSMCs) was tested using Dual-Luciferase Reporter System. SCAP(D443) mutant amplified from plasmid pTK-HSV-SCAP(D443N) and pSM22 from mice liver were cloned into pGL3-control vector to construct pGL3-SM22-SCAP(D443N) which was transfected into Chinese hamster ovary cells (CHO) to test SCAP(D443) expression by real-time PCR and Western blot. The sequence and construction of pGL3-SM22-SCAP(D443N) were correct. SM22 promoter activity initiated the expression of luciferase in VSMCs and also drove SCAP(D443) expression in transfected CHO cells. The pGL3-SM22-SCAP(D443N) eukaryotic expression vector was successfully constructed and the recombinant vector provides a powerful approach in investigating the function and regulation of SCAP and also in producing vascular smooth muscle specific SCAP transgenic mice.

  4. Intraclonal protein expression heterogeneity in recombinant CHO cells

    National Research Council Canada - National Science Library

    Pilbrough, Warren; Munro, Trent P; Gray, Peter

    2009-01-01

    .... Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers...

  5. Vectors

    DEFF Research Database (Denmark)

    Boeriis, Morten; van Leeuwen, Theo

    2017-01-01

    This article revisits the concept of vectors, which, in Kress and van Leeuwen’s Reading Images (2006), plays a crucial role in distinguishing between ‘narrative’, action-oriented processes and ‘conceptual’, state-oriented processes. The use of this concept in image analysis has usually focused...... on the most salient vectors, and this works well, but many images contain a plethora of vectors, which makes their structure quite different from the linguistic transitivity structures with which Kress and van Leeuwen have compared ‘narrative’ images. It can also be asked whether facial expression vectors...... should be taken into account in discussing ‘reactions’, which Kress and van Leeuwen link only to eyeline vectors. Finally, the question can be raised as to whether actions are always realized by vectors. Drawing on a re-reading of Rudolf Arnheim’s account of vectors, these issues are outlined...

  6. Synthesis, vibrational characteristic and luminescence properties of Er3+in HNH4ErP3O10

    Science.gov (United States)

    Chékir-Mzali, Jalila; Elgharbi, Sarra; Horchani-Naifer, Karima; Férid, Mokhtar

    2017-11-01

    Polycrystalline powders of the triphosphate of ammonium hydrogen and erbium NH4ErHP3O10 are grown by flux method. The obtained sample was characterized by X-Ray Diffraction, Infrared and Raman spectroscopies. It was found that this compound is isostructural with NH4BiHP3O10. It crystallizes in the triclinic system, with space group P-1. The spectroscopic properties have been investigated in the UV-Vis and IR region via absorption, excitation, and emission spectra at room temperature. Also, the particle size distribution was investigated and the average crystallite size of HNH4ErP3O10 was 124.08 nm. The optical band gap energy (Eg ≈ 4.24 eV) was calculated from the diffuse reflectance by using the Kubelka-Munk function and Tauc's relation. The time-resolved luminescence of 4F9/2 emission has also been studied. The decay time was shown to be exponential with a pure radiative transition and the lifetime value for the transition 4F9/2 → 4I15/2 is found equal to 15, 4 μs. The effects of the crystal structure, NH4+ and OH groups on the lifetime have been revealed. The CIE chromaticity coordinates reveal that this phosphor can be a promising Yellow-orange emitting material.

  7. Reactions of H+(pyridinem(H2On and H+(NH31(pyridinem(H2On with NH3: experiments and kinetic modelling

    Directory of Open Access Journals (Sweden)

    E. Uggerud

    2012-03-01

    Full Text Available Reactions between pyridine containing water cluster ions, H+(pyridine1(H2On, H+(pyridine2(H2On and H+(NH31(pyridine1(H2On (n up to 15 with NH3 have been studied experimentally using a quadrupole time-of-flight mass spectrometer. The product ions in the reaction between H+(pyridinem(H2On (m = 1 to 2 and NH3 have been determined for the first time. It is found that the reaction mainly leads to cluster ions of the form H+(NH31(pyridinem(H2On-x, with x = 1 or 2 depending on the initial size of the reacting cluster ion. For a given number of water molecules (from 5 to 15 in the cluster ion, rate coefficients are found to be slightly lower than those for protonated pure water clusters reacting with ammonia. The rate coefficients obtained from this study are used in a kinetic cluster ion model under tropospheric conditions. The disagreement between ambient ground level measurements and previous models are discussed in relation to the results from our model and future experimental directions are suggested.

  8. CHO Quasispecies—Implications for Manufacturing Processes

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    Florian M. Wurm

    2013-10-01

    Full Text Available Chinese hamster ovary (CHO cells are a source of multi-ton quantities of protein pharmaceuticals. They are, however, immortalized cells, characterized by a high degree of genetic and phenotypic diversity. As is known for any biological system, this diversity is enhanced by selective forces when laboratories (no sharing of gene pools grow cells under (diverse conditions that are practical and useful. CHO cells have been used in culture for more than 50 years, and various lines of cells are available and have been used in manufacturing. This article tries to represent, in a cursory way, the history of CHO cells, particularly the origin and subsequent fate of key cell lines. It is proposed that the name CHO represents many different cell types, based on their inherent genetic diversity and their dynamic rate of genetic change. The continuing remodeling of genomic structure in clonal or non-clonal cell populations, particularly due to the non-standardized culture conditions in hundreds of different labs renders CHO cells a typical case for “quasispecies”. This term was coined for families of related (genomic sequences exposed to high mutation rate environments where a large fraction of offspring is expected to carry one or more mutations. The implications of the quasispecies concept for CHO cells used in protein manufacturing processes are significant. CHO genomics/transcriptomics may provide only limited insights when done on one or two “old” and poorly characterized CHO strains. In contrast, screening of clonal cell lines, derived from a well-defined starting material, possibly within a given academic or industrial environment, may reveal a more narrow diversity of phenotypes with respect to physiological/metabolic activities and, thus, allow more precise and reliable predictions of the potential of a clone for high-yielding manufacturing processes.

  9. Dicty_cDB: CHO513 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO513 (Link to dictyBase) - - - Contig-U11723-1 CHO513P (Link... to Original site) CHO513F 451 CHO513Z 673 CHO513P 1104 - - Show CHO513 Library CH (Link to library) Clone ID CHO513 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U11723-1 Original site URL http://dict...SGLFTKKKGPYLXLCFDEDNSVKFQIEIVKICNLDLTGIQLKRLSGDTWKYKD ICTELVESMKL*vff*kknk***iikk**iinkikixnkikkk Translated...KKKGPYLXLCFDEDNSVKFQIEIVKICNLDLTGIQLKRLSGDTWKYKD ICTELVESMKL*vff*kknk***iikk**iinkikixnkikkk Homology vs CSM

  10. The art of CHO cell engineering: A comprehensive retrospect and future perspectives.

    Science.gov (United States)

    Fischer, Simon; Handrick, René; Otte, Kerstin

    2015-12-01

    Chinese hamster ovary (CHO) cells represent the most frequently applied host cell system for industrial manufacturing of recombinant protein therapeutics. CHO cells are capable of producing high quality biologics exhibiting human-like post-translational modifications in gram quantities. However, production processes for biopharmaceuticals using mammalian cells still suffer from cellular limitations such as limited growth, low productivity and stress resistance as well as higher expenses compared to bacterial or yeast based expression systems. Besides bioprocess, media and vector optimizations, advances in host cell engineering technologies comprising introduction, knock-out or post-transcriptional silencing of engineering genes have paved the way for remarkable achievements in CHO cell line development. Furthermore, thorough analysis of cellular pathways and mechanisms important for bioprocessing steadily unravels novel target molecules which might be addressed by functional genomic tools in order to establish superior production cell factories. This review provides a comprehensive summary of the most fundamental achievements in CHO cell engineering over the past three decades. Finally, the authors discuss the potential of novel and innovative methodologies that might contribute to further enhancement of existing CHO based production platforms for biopharmaceutical manufacturing in the future. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Synthesis of human prolactin in Chinese hamster ovary (CHO) cells; Sintese de prolactina humana em celulas de ovario de hamster chines (CHO)

    Energy Technology Data Exchange (ETDEWEB)

    Soares, Carlos Roberto Jorge

    2000-07-01

    Three different eukaryotic expression vectors, based on the same selectable gene marker (dhfr), have been used for dhf- CHO cells transfection to rapidly isolate stable cell lines capable of secreting high levels of recombinant human prolactin (rec-hPRL). Two vectors, one codifying a human prolactin (p658-hPRL) and the other a tag-prolactin (p658-tagPRL), contain the complete hepatitis B virus-X (HBV-X) gene coding for a viral transactivator and a sequence derived from the granulocyte-macrophage colony-stimulating factor (GM-CSF) that mediates selective dhfr mRNA degradation. These vectors have the advantage of rapidly obtaining stable cell lines without methotrexate amplification. The highest secretion obtained by these vectors was of approximately 10 {mu}g hPRU10{sup 6} cells/day. The other vector (pEDdc-hPRL) is based on a dicistronic expression system, containing an internal ribosome entry site isolated from the encephalomyocarditis (EMC) virus. This vector before amplification provided secretion levels at least 10 fold lower than that obtained with the other two vectors. However, after three steps of methotrexate amplification, it provided some clones able to secrete up to 30 {mu}g hPRU10{sup 6} cells/day. This is the first report describing the production and purification of rec-hPRL from CHO cells, obtaining secretion levels with both vectors higher than those reported so far for this hormone in other eukaryotic systems. CHO-derived rec-hPRL contained approximately 10 % of the glycosylated form, a value that is consistent with results reported for hPRL purified from the pituitary or from transformed murine C-127 cells. CHO-derived rec-hPRL was purified with good yield, obtaining also a good resolution between non-glycosylated and glycosylated prolactin. The latter, when its potency was determined via an in vitro bioassay, presented a 47 % lower bioactivity. A qualitative and quantitative analysis of these forms was also possible thanks to the setting up of a

  12. CHO-S antibody titers >1 gram/liter using flow electroporation-mediated transient gene expression followed by rapid migration to high-yield stable cell lines.

    Science.gov (United States)

    Steger, Krista; Brady, James; Wang, Weili; Duskin, Meg; Donato, Karen; Peshwa, Madhusudan

    2015-04-01

    In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TGE for gram-level production of antibodies without the need for specialized expression vectors or genetically engineered CHO cell lines. CHO cell flow electroporation is easily scaled from milligram to multigram quantities without protocol reoptimization while maintaining transfection performance and antibody productivity. In this article, data are presented that demonstrate the reproducibility, scalability, and antibody production capabilities of CHO-based TGE using the MaxCyte STX. Data show optimization of posttransfection parameters such as cell density, media composition, and feed strategy that result in secreted antibody titers >1 g/L and production of multiple grams of antibody within 2 weeks of a single CHO-S cell transfection. In addition, data are presented to demonstrate the application of scalable electroporation for the rapid generation of high-yield stable CHO cell lines to bridge the gap between early- and late-stage antibody development activities. © 2014 Society for Laboratory Automation and Screening.

  13. A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells.

    Science.gov (United States)

    Kang, Shin-Young; Kim, Yeon-Gu; Kang, Seunghee; Lee, Hong Weon; Lee, Eun Gyo

    2016-05-01

    Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics

    DEFF Research Database (Denmark)

    Kumar, Amit; Baycin-Hizal, Deniz; Wolozny, Daniel

    2015-01-01

    Chinese hamster ovary (CHO) cells are the preferred host cell line for manufacturing a variety of complex biotherapeutic drugs including monoclonal antibodies. We performed a proteomics and bioinformatics analysis on the spent medium from adherent CHO cells. Supernatant from CHO-K1 culture...... was collected and subjected to in-solution digestion followed by LC/LC-MS/MS analysis, which allowed the identification of 3281 different host cell proteins (HCPs). To functionally categorize them, we applied multiple bioinformatics tools to the proteins identified in our study including SignalP, Target......P, SecretomeP, TMHMM, WoLF PSORT, and Phobius. This analysis provided information on the presence of signal peptides, transmembrane domains, and cellular localization and showed that both secreted and intracellular proteins were constituents of the supernatant. Identified proteins were shown to be localized...

  15. Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics

    Science.gov (United States)

    Kumar, Amit; Baycin-Hizal, Deniz; Wolozny, Daniel; Pedersen, Lasse Ebdrup; Lewis, Nathan E.; Heffner, Kelley; Chaerkady, Raghothama; Cole, Robert N.; Shiloach, Joseph; Zhang, Hui; Bowen, Michael A.; Betenbaugh, Michael J.

    2016-01-01

    Chinese hamster ovary (CHO) cells are the preferred host cell line for manufacturing a variety of complex biotherapeutic drugs including monoclonal antibodies. We performed a proteomics and bioinformatics analysis on the spent medium from adherent CHO cells. Supernatant from CHO-K1 culture was collected and subjected to in-solution digestion followed by LC/LC–MS/MS analysis, which allowed the identification of 3281 different host cell proteins (HCPs). To functionally categorize them, we applied multiple bioinformatics tools to the proteins identified in our study including SignalP, TargetP, SecretomeP, TMHMM, WoLF PSORT, and Phobius. This analysis provided information on the presence of signal peptides, transmembrane domains, and cellular localization and showed that both secreted and intracellular proteins were constituents of the supernatant. Identified proteins were shown to be localized to the secretory pathway including ones playing roles in cell growth, proliferation, and folding as well as those involved in protein degradation and removal. After combining proteins predicted to be secreted or having a signal peptide, we identified 1015 proteins, which we termed as CHO supernatant-ome (CHO-SO), or superome. As a part of this effort, we created a publically accessible web-based tool called GO–CHO to functionally categorize proteins found in CHO-SO and identify enriched molecular functions, biological processes, and cellular components. We also used a tool to evaluate the immunogenicity potential of high-abundance HCPs. Among enriched functions were catalytic activity and structural constituents of the cytoskeleton. Various transport related biological processes, such as vesicle mediated transport, were found to be highly enriched. Extracellular space and vesicular exosome associated proteins were found to be the most enriched cellular components. The superome also contained proteins secreted from both classical and nonclassical secretory pathways. The work

  16. A high density CHO-S transient transfection system: Comparison of ExpiCHO and Expi293.

    Science.gov (United States)

    Jain, Nina K; Barkowski-Clark, Susan; Altman, Richard; Johnson, Krista; Sun, Fang; Zmuda, Jonathan; Liu, Chao Yan; Kita, Adriana; Schulz, Ryan; Neill, Alyssa; Ballinger, Robert; Patel, Rekha; Liu, Jian; Mpanda, Alinafe; Huta, Brian; Chiou, Henry; Voegtli, Walter; Panavas, Tadas

    2017-06-01

    Chinese Hamster Ovary (CHO) cells are the principal mammalian host used for stable cell line generation and biotherapeutic protein production. Until recently, production of milligrams to grams of protein in CHO transient systems was challenging. As such, Human Embryonic Kidney (HEK293) cells are the most common mammalian cell type used for transient transfection. The post-translational modifications (PTMs) of a protein are dictated in part by the cell line used for expression, and changes in PTMs have been shown to affect both the activity and biophysical properties of proteins. Therefore, it is potentially advantageous to keep the host cell type consistent throughout drug discovery and development. To this end, we compared the ExpiCHO system, a high density CHO-S transient transfection system, to the Expi293 and FreeStyle MAX CHO transient systems. Fourteen proteins were expressed in both the Expi293 and ExpiCHO systems. For a majority of proteins tested, the protein titers observed with the ExpiCHO system were higher than those seen with both the FreeStyle MAX CHO and Expi293 systems. Antibodies expressed using the ExpiCHO system had glycosylation patterns more similar to antibodies produced in stable CHO cell lines than Expi293-derived antibodies. However, culture duration and temperature were found to affect protein titer, monodispersity, enzyme activity, and PTMs and should be carefully selected when using the ExpiCHO system. The ExpiCHO transient transfection systems allows for facile production of milligrams to grams of protein in CHO cells and de-risks the transition from transient to stable material during drug development. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Intraclonal protein expression heterogeneity in recombinant CHO cells.

    Science.gov (United States)

    Pilbrough, Warren; Munro, Trent P; Gray, Peter

    2009-12-23

    Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean), approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations). Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50 days. Noise

  18. Intraclonal protein expression heterogeneity in recombinant CHO cells.

    Directory of Open Access Journals (Sweden)

    Warren Pilbrough

    Full Text Available Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean, approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations. Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50

  19. The Products of the Thermal Decomposition of CH3CHO

    Energy Technology Data Exchange (ETDEWEB)

    Vasiliou, AnGayle; Piech, Krzysztof M.; Zhang, Xu; Nimlos, Mark R.; Ahmed, Musahid; Golan, Amir; Kostko, Oleg; Osborn, David L.; Daily, John W.; Stanton, John F.; Ellison, G. Barney

    2011-04-06

    We have used a heated 2 cm x 1 mm SiC microtubular (mu tubular) reactor to decompose acetaldehyde: CH3CHO + DELTA --> products. Thermal decomposition is followed at pressures of 75 - 150 Torr and at temperatures up to 1700 K, conditions that correspond to residence times of roughly 50 - 100 mu sec in the mu tubular reactor. The acetaldehyde decomposition products are identified by two independent techniques: VUV photoionization mass spectroscopy (PIMS) and infrared (IR) absorption spectroscopy after isolation in a cryogenic matrix. Besides CH3CHO, we have studied three isotopologues, CH3CDO, CD3CHO, and CD3CDO. We have identified the thermal decomposition products CH3(PIMS), CO (IR, PIMS), H (PIMS), H2 (PIMS), CH2CO (IR, PIMS), CH2=CHOH (IR, PIMS), H2O (IR, PIMS), and HC=CH (IR, PIMS). Plausible evidence has been found to support the idea that there are at least three different thermal decomposition pathways for CH3CHO: Radical decomposition: CH3CHO + DELTA --> CH3 + [HCO] --> CH3 + H + CO Elimination: CH3CHO + DELTA --> H2 + CH2=C=O. Isomerization/elimination: CH3CHO + DELTA --> [CH2=CH-OH] --> HC=CH + H2O. Both PIMS and IR spectroscopy show compelling evidence for the participation of vinylidene, CH2=C:, as an intermediate in the decomposition of vinyl alchohol: CH2=CH-OH + DELTA --> [CH2=C:] + H2O --> HC=CH + H2O.

  20. Methods for modeling chinese hamster ovary (cho) cell metabolism

    DEFF Research Database (Denmark)

    2015-01-01

    Embodiments of the present invention generally relate to the computational analysis and characterization biological networks at the cellular level in Chinese Hamster Ovary (CHO) cells. Based on computational methods utilizing a hamster reference genome, the invention provides methods...

  1. Glycoengineering in CHO cells: Advances in systems biology

    DEFF Research Database (Denmark)

    Tejwani, Vijay; Andersen, Mikael Rørdam; Nam, Jong Hyun

    2018-01-01

    For several decades, glycoprotein biologics have been successfully produced from Chinese hamster ovary (CHO) cells. The therapeutic efficacy and potency of glycoprotein biologics are often dictated by their post translational modifications, particularly glycosylation, which unlike protein synthesis...

  2. Engineered CHO cells for production of diverse, homogeneous glycoproteins

    DEFF Research Database (Denmark)

    Yang, Zhang; Wang, Shengjun; Halim, Adnan

    2015-01-01

    Production of glycoprotein therapeutics in Chinese hamster ovary (CHO) cells is limited by the cells' generic capacity for N-glycosylation, and production of glycoproteins with desirable homogeneous glycoforms remains a challenge. We conducted a comprehensive knockout screen of glycosyltransferase...

  3. [GFP reporter gene under the direction of chicken ovalbumin gene promoter expressed in the CHO cell and in the primary cell cultures of chicken oviduct].

    Science.gov (United States)

    Pang, Yue; Li, Qing-Wei

    2005-01-01

    To reseach GFP reporter gene under the control of chick ovalbumin gene regulatory elements express in the CHO cell and in the primary cell cultures of chicken oviduct. 1.5kb fragment and 2.9kb fragment were amplicated by PCR method, two fragments were subeloned to manmalian expression vector pGFP-N2 by recombinant DNA technology, the CMV promoter was cut off from pGFP-N2, so two expression vectors were constructed, one is the P2.9koval-GFP including promoter, first exon, first intron of chicken ovalbumin gene, the other is the P1.5koval-GFP including first intron of chicken ovalbumin gene. Restriction enzyme digestion and DNA sequence analysis revealed that 5'upstream regions of ovalbumin gene were not only identical to those of the published chicken ovalbumin gene, but also were contained in the recombinant vector. They were transfected into the CHO cell and the primary cell cultures of chicken oviduct by Lipofectin, they were used for fluorescence detection. GFP protein existed in GFP transfected the CHO cell and the primary cell cultures of chicken oviduct. It is demonstrated that GFP reporter gene under the direction of chick ovalbumin gene promoter could be expressed in the CHO cell and in the primary cell cultures of chicken oviduct.

  4. Ubiquitous Chromatin Opening Elements (UCOEs) effect on transgene position and expression stability in CHO cells following methotrexate (MTX) amplification.

    Science.gov (United States)

    Betts, Zeynep; Dickson, Alan J

    2016-03-01

    The requirement for complex therapeutic proteins has resulted in mammalian cells, especially CHO cells, being the dominant host for recombinant protein manufacturing. In creating recombinant CHO cell lines, the expression vectors integrate into various parts of the genome leading to variable levels of expression and stability of protein production. This makes mammalian cell line development a long and laborious process. Therefore, with the intention to accelerate process development of recombinant protein production in CHO systems, UCOEs are utilized to diminish instability of production by maintaining an open chromatin surrounding in combination with MTX amplification. Chromosome painting and FISH analysis were performed to provide detailed molecular evaluation on the location of amplified genes and its relationship to the productivity and stability of the amplified cell lines. In summary, cell lines generated with vectors containing UCOEs retained stable GFP expression with MTX present (but instability was observed in the absence of MTX). UCOE cell lines displayed a higher frequency of integration into >1 chromosome than non-UCOE group. Cell populations were more homogenous in terms of transgene location at the end of Long-term culture (LTC). Overall our findings suggest variation in eGFP fluorescence may be attributed to changes in transgene integration profile over LTC. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. The products of the thermal decomposition of CH3CHO.

    Science.gov (United States)

    Vasiliou, AnGayle; Piech, Krzysztof M; Zhang, Xu; Nimlos, Mark R; Ahmed, Musahid; Golan, Amir; Kostko, Oleg; Osborn, David L; Daily, John W; Stanton, John F; Ellison, G Barney

    2011-07-07

    We have used a heated 2 cm × 1 mm SiC microtubular (μtubular) reactor to decompose acetaldehyde: CH(3)CHO + Δ → products. Thermal decomposition is followed at pressures of 75-150 Torr and at temperatures up to 1675 K, conditions that correspond to residence times of roughly 50-100 μs in the μtubular reactor. The acetaldehyde decomposition products are identified by two independent techniques: vacuum ultraviolet photoionization mass spectroscopy (PIMS) and infrared (IR) absorption spectroscopy after isolation in a cryogenic matrix. Besides CH(3)CHO, we have studied three isotopologues, CH(3)CDO, CD(3)CHO, and CD(3)CDO. We have identified the thermal decomposition products CH(3) (PIMS), CO (IR, PIMS), H (PIMS), H(2) (PIMS), CH(2)CO (IR, PIMS), CH(2)=CHOH (IR, PIMS), H(2)O (IR, PIMS), and HC≡CH (IR, PIMS). Plausible evidence has been found to support the idea that there are at least three different thermal decomposition pathways for CH(3)CHO; namely, radical decomposition: CH(3)CHO + Δ → CH(3) + [HCO] → CH(3) + H + CO; elimination: CH(3)CHO + Δ → H(2) + CH(2)=C=O; isomerization∕elimination: CH(3)CHO + Δ → [CH(2)=CH-OH] → HC≡CH + H(2)O. An interesting result is that both PIMS and IR spectroscopy show compelling evidence for the participation of vinylidene, CH(2)=C:, as an intermediate in the decomposition of vinyl alcohol: CH(2)=CH-OH + Δ → [CH(2)=C:] + H(2)O → HC≡CH + H(2)O.

  6. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

    Directory of Open Access Journals (Sweden)

    Tetsushi Sakuma

    2015-10-01

    Full Text Available Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc gene, in Chinese hamster ovary (CHO cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

  7. Identification of a novel temperature sensitive promoter in cho cells

    Directory of Open Access Journals (Sweden)

    Hesse Friedemann

    2011-05-01

    Full Text Available Abstract Background The Chinese hamster ovary (CHO expression system is the leading production platform for manufacturing biopharmaceuticals for the treatment of numerous human diseases. Efforts to optimize the production process also include the genetic construct encoding the therapeutic gene. Here we report about the successful identification of an endogenous highly active gene promoter obtained from CHO cells which shows conditionally inducible gene expression at reduced temperature. Results Based on CHO microarray expression data abundantly transcribed genes were selected as potential promoter candidates. The S100a6 (calcyclin and its flanking regions were identified from a genomic CHO-K1 lambda-phage library. Computational analyses showed a predicted TSS, a TATA-box and several TFBSs within the 1.5 kb region upstream the ATG start signal. Various constructs were investigated for promoter activity at 37°C and 33°C in transient luciferase reporter gene assays. Most constructs showed expression levels even higher than the SV40 control and on average a more than two-fold increase at lower temperature. We identified the core promoter sequence (222 bp comprising two SP1 sites and could show a further increase in activity by duplication of this minimal sequence. Conclusions This novel CHO promoter permits conditionally high-level gene expression. Upon a shift to 33°C, a two to three-fold increase of basal productivity (already higher than SV40 promoter is achieved. This property is of particular advantage for a process with reduced expression during initial cell growth followed by the production phase at low temperature with a boost in expression. Additionally, production of toxic proteins becomes feasible, since cell metabolism and gene expression do not directly interfere. The CHO S100a6 promoter can be characterized as cold-shock responsive with the potential for improving process performance of mammalian expression systems.

  8. Metabolite profiling of CHO cells: Molecular reflections of bioprocessing effectiveness

    NARCIS (Netherlands)

    Sellick, C.A.; Croxford, A.S.; Maqsood, A.R.; Stephens, G.M.; Westerhoff, H.V.; Goodacre, R.; Dickson, A.J.

    2015-01-01

    Whilst development of medium and feeds has provided major advances in recombinant protein production in CHO cells, the fundamental understanding is limited. We have applied metabolite profiling with established robust (GC-MS) analytics to define the molecular loci by which two yield-enhancing feeds

  9. Perforate on CHO cell membranes induced by electromagnetic ...

    African Journals Online (AJOL)

    Perforate on CHO cell membranes induced by electromagnetic pulses irradiation observed by atomic force microscopy. ... increased with an increase in applied EMP field intensity and an increase in the number of EMP pulses. EMP induced membrane perforate may play a very important role in EMP biological effects.

  10. A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær

    2014-01-01

    A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique – USER cloning – to rapidly construct mammalian expression vectors of multiple DNA fragments...... efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells......, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors....

  11. Lysophosphatidic acid activates peroxisome proliferator activated receptor-γ in CHO cells that over-express glycerol 3-phosphate acyltransferase-1.

    Directory of Open Access Journals (Sweden)

    Cliona M Stapleton

    Full Text Available Lysophosphatidic acid (LPA is an agonist for peroxisome proliferator activated receptor-γ (PPARγ. Although glycerol-3-phosphate acyltransferase-1 (GPAT1 esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression.

  12. Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells.

    Science.gov (United States)

    Wang, X Y; Zhang, J H; Sun, Q L; Yao, Z Y; Deng, B G; Guo, W Y; Wang, L; Dong, W H; Wang, F; Zhao, C P; Wang, T Y

    2015-08-07

    Preliminary studies have suggested that a characteristic element of the matrix attachment region (MAR) in human interferon-β mediates the adhesion of vectors to Chinese hamster ovary (CHO) cells. In this study, we investigated if vector adhesion increased nerve growth factor (NGF) expression in CHO cells. The MAR characteristic element sequence of human interferon-β was inserted into the multiple-cloning site of the pEGFP-C1 vector. The target NGF gene was inserted upstream of the MAR characteristic element sequence to construct the MAR/NGF expression vector. The recombinant plasmid was transfected into CHO cells and stable monoclonal cells were selected using G418. NGF mRNA and protein expression was detected by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Plasmid reduction experiments were used to determine the state of transfected plasmid in mammalian cells. The insertion of MAR into the vector increased NGF expression levels in CHO cells (1.93- fold) compared to the control. The recombinant plasmid expressing the MAR sequence was digested into a linear space vector. The inserted MAR and NGF sequences were consistent with those inserted into the plasmid before recombination. Therefore, we concluded that the MAR characteristic element mediates vector adhesion to CHO cells and enhances the stability and efficiency of the target gene expression.

  13. Choline Uptake in Agrobacterium tumefaciens by the High-Affinity ChoXWV Transporter▿

    Science.gov (United States)

    Aktas, Meriyem; Jost, Kathinka A.; Fritz, Christiane; Narberhaus, Franz

    2011-01-01

    Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called “Venus flytrap mechanism” of substrate binding. PMID:21803998

  14. Genotoxicity of complex mixtures: CHO cell mutagenicity assay

    Energy Technology Data Exchange (ETDEWEB)

    Frazier, M.E.; Samuel, J.E.

    1985-02-01

    A Chinese hamster ovary (CHO) mammalian cell assay was used to evaluate the genotoxicity of complex mixtures (synthetic fuels). The genotoxicity (mutagenic potency) of the mixtures increased as the temperature of their boiling range increased. Most of the genotoxicity in the 750/sup 0/F+ boiling-range materials was associated with the neutral polycyclic aromatic hydrocarbon (PAH) fractions. Chemical analysis data indicate that the PAH fractions of high-boiling coal liquids contain a number of known chemical carcinogens, including five- and six-ring polyaromatics (e.g., benzo(a)pyrene) as well as four- and five-ring alkyl-substituted PAH (e.g., methylchrysene and dimethylbenzanthracenes); concentrations are a function of boiling point (bp). In vitro genotoxicity was also detected in fractions of nitrogen-containing polyaromatic compounds, as well as in those with aliphatics of hydroxy-containing PAH. Mutagenic activity of some fractions was detectable in the CHO assay in the absence of an exogenous metabolic activation system; in some instances, addition of exogenous enzymes and cofactors inhibited expression of the direct-acting mutagenic potential of the fraction. These data indicate that the organic matrix of the chemical fraction determines whether, and to what degree, various mutagens are expressed in the CHO assay. Therefore, the results of biological assays of these mixtures must be correlated with chemical analyses for proper interpretation of these data. 29 references, 16 figures, 4 tables.

  15. About vectors

    CERN Document Server

    Hoffmann, Banesh

    1975-01-01

    From his unusual beginning in ""Defining a vector"" to his final comments on ""What then is a vector?"" author Banesh Hoffmann has written a book that is provocative and unconventional. In his emphasis on the unresolved issue of defining a vector, Hoffmann mixes pure and applied mathematics without using calculus. The result is a treatment that can serve as a supplement and corrective to textbooks, as well as collateral reading in all courses that deal with vectors. Major topics include vectors and the parallelogram law; algebraic notation and basic ideas; vector algebra; scalars and scalar p

  16. Vector analysis

    CERN Document Server

    Newell, Homer E

    2006-01-01

    When employed with skill and understanding, vector analysis can be a practical and powerful tool. This text develops the algebra and calculus of vectors in a manner useful to physicists and engineers. Numerous exercises (with answers) not only provide practice in manipulation but also help establish students' physical and geometric intuition in regard to vectors and vector concepts.Part I, the basic portion of the text, consists of a thorough treatment of vector algebra and the vector calculus. Part II presents the illustrative matter, demonstrating applications to kinematics, mechanics, and e

  17. Characterization of Host Cell Proteins (HCPs) in CHO Cell Bioprocesses.

    Science.gov (United States)

    Hogwood, Catherine E M; Chiverton, Lesley M; Mark Smales, C

    2017-01-01

    Host cell protein content during bioprocessing of biotherapeutic proteins generated from cultured Chinese hamster ovary (CHO) cells is typically measured using immunological and gel-based methods. Estimation of HCP concentration is usually undertaken using Enzyme-Linked ImmunoSorbent Assays (ELISA), while estimation of HCP clearance/presence can be achieved by comparing 2D-PAGE images of samples and by undertaking western blotting of 2D-PAGE analyzed samples. Here, we describe the analyses of HCP content using these methodologies.

  18. Elementary vectors

    CERN Document Server

    Wolstenholme, E Œ

    1978-01-01

    Elementary Vectors, Third Edition serves as an introductory course in vector analysis and is intended to present the theoretical and application aspects of vectors. The book covers topics that rigorously explain and provide definitions, principles, equations, and methods in vector analysis. Applications of vector methods to simple kinematical and dynamical problems; central forces and orbits; and solutions to geometrical problems are discussed as well. This edition of the text also provides an appendix, intended for students, which the author hopes to bridge the gap between theory and appl

  19. High-CHO diet increases post-exercise oxygen consumption after a supramaximal exercise bout

    Science.gov (United States)

    Ferreira, G.A.; Bertuzzi, R.; De-Oliveira, F.R.; Pires, F.O.; Lima-Silva, A.E.

    2016-01-01

    We investigated if carbohydrate (CHO) availability could affect the excess post-exercise oxygen consumption (EPOC) after a single supramaximal exercise bout. Five physically active men cycled at 115% of peak oxygen uptake (V̇O2 peak) until exhaustion with low or high pre-exercise CHO availability. The endogenous CHO stores were manipulated by performing a glycogen-depletion exercise protocol 48 h before the trial, followed by 48 h consuming either a low- (10% CHO) or a high-CHO (80% CHO) diet regime. Compared to the low-CHO diet, the high-CHO diet increased time to exhaustion (3.0±0.6 min vs 4.4±0.6, respectively, P=0.01) and the total O2 consumption during the exercise (6.9±0.9 L and 11.3±2.1, respectively, P=0.01). This was accompanied by a higher EPOC magnitude (4.6±1.8 L vs 6.2±2.8, respectively, P=0.03) and a greater total O2 consumption throughout the session (exercise+recovery: 11.5±2.5 L vs 17.5±4.2, respectively, P=0.01). These results suggest that a single bout of supramaximal exercise performed with high CHO availability increases both exercise and post-exercise energy expenditure. PMID:27783812

  20. Vector analysis

    CERN Document Server

    Brand, Louis

    2006-01-01

    The use of vectors not only simplifies treatments of differential geometry, mechanics, hydrodynamics, and electrodynamics, but also makes mathematical and physical concepts more tangible and easy to grasp. This text for undergraduates was designed as a short introductory course to give students the tools of vector algebra and calculus, as well as a brief glimpse into these subjects' manifold applications. The applications are developed to the extent that the uses of the potential function, both scalar and vector, are fully illustrated. Moreover, the basic postulates of vector analysis are brou

  1. RNA-seq based expression analysis of the CHO cell protein secretion pathway

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kaas, Christian Schrøder; Kildegaard, Helene Faustrup

    The Chinese hamster ovary (CHO) cell-line is the predominant mammalian industrial cell line being used to produce recombinant therapeutic proteins. Although CHO cells have been used for more than 25 years, the genome sequence was first published in 2011. So far there have been limited studies...

  2. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

    DEFF Research Database (Denmark)

    Xu, Xun; Pan, Shengkai; Liu, Xin

    2011-01-01

    Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most...

  3. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

    DEFF Research Database (Denmark)

    Xu, Xun; Pan, Shengkai; Liu, Xin

    2011-01-01

    Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of...

  4. Efficiency of gene transfection reagents in NG108-15, SH-SY5Y and CHO-K1 cell lines.

    Science.gov (United States)

    Martín-Montañez, E; López-Téllez, J F; Acevedo, M J; Pavía, J; Khan, Z U

    2010-06-01

    Several gene delivery reagents were analyzed for their transfection efficiency. Genes studied belonged to the class of mammalian proteins termed regulators of G-protein signaling (RGS), ranged in size up to 2.2 Kb long and were transfected into the NG108-15, SH-SY5Y and CHO-K1 cell lines. Prior to transfection, genes were cloned into a nonviral vector pcDNA 6.2/EmGFP, so as to express a green fluorescent protein tag at the 3' end. Flow cytometry was used to analyze cell fluorescent activity and thereby transfection efficiency. Gene delivery reagents Lipofectamine 2000 and ExGen 500 produced more effective transfection in NG108-15 cells whereas Lipofectamine 2000, ExGen 500 and TurboFectin 8.0 were more effective in CHO-K1 cells. In both these cell lines, transfection efficiency reached 60-80%. In SH-SY5Y cells, TurboFectin 8.0 produced the best transfection result; however efficiency level was only 5%. Gene size had no effect on transfection efficiency. Unlike Lipofectamine 2000, cells transfected using ExGen 500 showed morphological deformation. Our results suggest that Lipofectamine 2000 is the most suitable transfection medium for gene delivery to NG108-15 and CHO-K1 cells. Copyright 2010 Prous Science, S.A.U. or its licensors. All rights reserved.

  5. Karen Resistance Poetry translated and introduced by Violet Cho

    Directory of Open Access Journals (Sweden)

    Violet Cho

    2014-05-01

    Full Text Available Karen Resistance Poetry translated and introduced by Violet Cho. Tee Noe was born as M. No Noe in a village called Thavorta, Karen State, Myanmar (Burma in 1952. After completing year 10 at a state high school in 1974, he worked as a junior clerk at a local government office in Karen State, eastern Myanmar. Later he joined the rebellion as a soldier for the Karen National Liberation Army and as a schoolteacher in Burmese refugee camps along Thai-Burma border. With no formal knowledge of the mechanics of poetry, Tee Noe has become a leading voice of the Karen diaspora. From a young age, Noe was drawn to poetry. He remembers singing a short hta (Karen oral poem to thank his cousin who gave him a woollen hat as a present when he turned six: 'To school I run when the bell rings, with a woollen hat today I went.' "

  6. Cloning vector

    Science.gov (United States)

    Guilfoyle, R.A.; Smith, L.M.

    1994-12-27

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site. 2 figures.

  7. Cloning vector

    Science.gov (United States)

    Guilfoyle, Richard A.; Smith, Lloyd M.

    1994-01-01

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site.

  8. CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycan profiles

    DEFF Research Database (Denmark)

    Amann, Thomas; Hansen, Anders Holmgaard; Pristovsek, Nusa

    Combining the chinese hamster ovary (CHO) - K1 draft genome1,2, identified CHO glycosyltransferases3 and the power of multiplexing gene knock-outs with CRISPR/Cas94 via co-transfection of Cas9 and one single guiding RNA (sgRNA) per target, we generated 20 Rituximab expressing CHO-S cell lines...

  9. CRISPR/Cas9-mediated genome engineering of CHO cell factories: Application and perspectives.

    Science.gov (United States)

    Lee, Jae Seong; Grav, Lise Marie; Lewis, Nathan E; Faustrup Kildegaard, Helene

    2015-07-01

    Chinese hamster ovary (CHO) cells are the most widely used production host for therapeutic proteins. With the recent emergence of CHO genome sequences, CHO cell line engineering has taken on a new aspect through targeted genome editing. The bacterial clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables rapid, easy and efficient engineering of mammalian genomes. It has a wide range of applications from modification of individual genes to genome-wide screening or regulation of genes. Facile genome editing using CRISPR/Cas9 empowers researchers in the CHO community to elucidate the mechanistic basis behind high level production of proteins and product quality attributes of interest. In this review, we describe the basis of CRISPR/Cas9-mediated genome editing and its application for development of next generation CHO cell factories while highlighting both future perspectives and challenges. As one of the main drivers for the CHO systems biology era, genome engineering with CRISPR/Cas9 will pave the way for rational design of CHO cell factories. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. The emerging CHO systems biology era: harnessing the ‘omics revolution for biotechnology

    DEFF Research Database (Denmark)

    Kildegaard, Helene Faustrup; Baycin-Hizal, Deniz; Lewis, Nathan

    2013-01-01

    line was recently sequenced. Now, the CHO systems biology era is underway. Critical ‘omics data sets, including proteomics, transcriptomics, metabolomics, fluxomics, and glycomics, are emerging, allowing the elucidation of the molecular basis of CHO cell physiology. The incorporation of these data sets...... into mathematical models that describe CHO phenotypes will provide crucial biotechnology insights. As ‘omics technologies and computational systems biology mature, genome-scale approaches will lead to major innovations in cell line development and metabolic engineering, thereby improving protein production...

  11. CRISPR/Cas9-mediated genome engineering of CHO cell factories: application and perspectives

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Grav, Lise Marie; Lewis, Nathan E.

    2015-01-01

    repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables rapid,easy and efficient engineering of mammalian genomes. It has a wide range of applications frommodification of individual genes to genome-wide screening or regulation of genes. Facile genomeediting using CRISPR/Cas9 empowers...... researchers in the CHO community to elucidate the mechanisticbasis behind high level production of proteins and product quality attributes of interest. Inthis review, we describe the basis of CRISPR/Cas9-mediated genome editing and its applicationfor development of next generation CHO cell factories while...... highlighting both future perspectivesand challenges. As one of the main drivers for the CHO systems biology era, genome engineeringwith CRISPR/Cas9 will pave the way for rational design of CHO cell factories....

  12. CHOmine: an integrated data warehouse for CHO systems biology and modeling.

    Science.gov (United States)

    Gerstl, Matthias P; Hanscho, Michael; Ruckerbauer, David E; Zanghellini, Jürgen; Borth, Nicole

    2017-01-01

    The last decade has seen a surge in published genome-scale information for Chinese hamster ovary (CHO) cells, which are the main production vehicles for therapeutic proteins. While a single access point is available at www.CHOgenome.org, the primary data is distributed over several databases at different institutions. Currently research is frequently hampered by a plethora of gene names and IDs that vary between published draft genomes and databases making systems biology analyses cumbersome and elaborate. Here we present CHOmine, an integrative data warehouse connecting data from various databases and links to other ones. Furthermore, we introduce CHOmodel, a web based resource that provides access to recently published CHO cell line specific metabolic reconstructions. Both resources allow to query CHO relevant data, find interconnections between different types of data and thus provides a simple, standardized entry point to the world of CHO systems biology. http://www.chogenome.org.

  13. Utilization and evaluation of CHO-specific sequence databases for mass spectrometry based proteomics.

    Science.gov (United States)

    Meleady, Paula; Hoffrogge, Raimund; Henry, Michael; Rupp, Oliver; Bort, Juan Hernandez; Clarke, Colin; Brinkrolf, Karina; Kelly, Shane; Müller, Benjamin; Doolan, Padraig; Hackl, Matthias; Beckmann, Tim Frederik; Noll, Thomas; Grillari, Johannes; Barron, Niall; Pühler, Alf; Clynes, Martin; Borth, Nicole

    2012-06-01

    Recently released sequence information on Chinese hamster ovary (CHO) cells promises to not only facilitate our understanding of these industrially important cell factories through direct analysis of the sequence, but also to enhance existing methodologies and allow new tools to be developed. In this article we demonstrate the utilization of CHO specific sequence information to improve mass spectrometry (MS) based proteomic identification. The use of various CHO specific databases enabled the identification of 282 additional proteins, thus increasing the total number of identified proteins by 40-50%, depending on the sample source and methods used. In addition, a considerable portion of those proteins that were identified previously based on inter-species sequence homology were now identified by a larger number of peptides matched, thus increasing the confidence of identification. The new sequence information offers improved interpretation of proteomic analyses and will, in the years to come, prove vital to unraveling the CHO proteome. Copyright © 2012 Wiley Periodicals, Inc.

  14. Metabolite profiling of recombinant CHO cells: designing tailored feeding regimes that enhance recombinant antibody production.

    NARCIS (Netherlands)

    Sellick, C.A.; Croxford, A.S.; Maqsood, A.R.; Stephens, G.; Westerhoff, H.V.; Goodacre, R.; Dickson, A.J.

    2011-01-01

    Chinese hamster ovary (CHO) cells are the primary platform for commercial expression of recombinant therapeutic proteins. Obtaining maximum production from the expression platform requires optimal cell culture medium (and associated nutrient feeds). We have used metabolite profiling to define the

  15. Metabolite profiling of recombinant CHO cells: Designing tailored feeding regimes that enhance recombinant antibody production.

    NARCIS (Netherlands)

    Sellick, C.A.; Croxford, A.S.; Maqsood, A.R.; Stephens, G.; Westerhoff, H.V.; Goodacre, R.; Dickson, A.J.

    2011-01-01

    Chinese hamster ovary (CHO) cells are the primary platform for commercial expression of recombinant therapeutic proteins. Obtaining maximum production from the expression platform requires optimal cell culture medium (and associated nutrient feeds). We have used metabolite profiling to define the

  16. Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells.

    Directory of Open Access Journals (Sweden)

    Saeedeh Ebadat

    Full Text Available In recent years, monoclonal antibodies (mAbs have been developed as powerful therapeutic and diagnostic agents and Chinese hamster ovary (CHO cells have emerged as the dominant host for the recombinant expression of these proteins. A critical step in recombinant expression is the utilization of strong promoters, such as the Chinese Hamster Elongation Factor-1α (CHEF-1 promoter. To compare the strengths of CHEF with cytomegalovirus (CMV promoter for mAb expression in CHO cells, four bicistronic vectors bearing either internal ribosome entry site (IRES or Furin/2A (F2A sequences were designed. The efficiency of these promoters was evaluated by measuring level of expressed antibody in stable cell pools. Our results indicated that CHEF promoter-based expression of mAbs was 2.5 fold higher than CMV-based expression in F2A-mediated vectors. However, this difference was less significant in IRES-mediated mAb expressing cells. Studying the stability of the F2A expression system in the course of 18 weeks, we observed that the cells having CHEF promoter maintained their antibody expression at higher level than those transfected with CMV promoter. Further analyses showed that both IRES-mediated vectors, expressed mAbs with correct size, whereas in antibodies expressed via F2A system heterogeneity of light chains were detected due to incomplete furin cleavage. Our findings indicated that the CHEF promoter is a viable alternative to CMV promoter-based expression in F2A-mediated vectors by providing both higher expression and level of stability.

  17. Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression.

    Science.gov (United States)

    Lin, Nan; Mascarenhas, Joaquina; Sealover, Natalie R; George, Henry J; Brooks, Jeanne; Kayser, Kevin J; Gau, Brian; Yasa, Isil; Azadi, Parastoo; Archer-Hartmann, Stephanie

    2015-01-01

    N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN(®) GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones ("ST6GAL1 OE Clone 31 and 32") were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of "bio-better" protein therapeutics and cell culture vaccine production. © 2015 American Institute of Chemical Engineers.

  18. Fed-Batch CHO Cell Culture for Lab-Scale Antibody Production.

    Science.gov (United States)

    Fan, Yuzhou; Ley, Daniel; Andersen, Mikael Rørdam

    2018-01-01

    Fed-batch culture is the most commonly used upstream process in industry today for recombinant monoclonal antibody production using Chinese hamster ovary (CHO) cells. Developing and optimizing this process in the lab is crucial for establishing process knowledge, which enables rapid and predictable tech-transfer to manufacturing scale. In this chapter, we describe stepwise how to carry out fed-batch CHO cell culture for lab-scale antibody production.

  19. Vector geometry

    CERN Document Server

    Robinson, Gilbert de B

    2011-01-01

    This brief undergraduate-level text by a prominent Cambridge-educated mathematician explores the relationship between algebra and geometry. An elementary course in plane geometry is the sole requirement for Gilbert de B. Robinson's text, which is the result of several years of teaching and learning the most effective methods from discussions with students. Topics include lines and planes, determinants and linear equations, matrices, groups and linear transformations, and vectors and vector spaces. Additional subjects range from conics and quadrics to homogeneous coordinates and projective geom

  20. VECTOR INTEGRATION

    NARCIS (Netherlands)

    Thomas, E. G. F.

    2012-01-01

    This paper deals with the theory of integration of scalar functions with respect to a measure with values in a, not necessarily locally convex, topological vector space. It focuses on the extension of such integrals from bounded measurable functions to the class of integrable functions, proving

  1. Reaction of Cl atoms with C6F13CH2OH, C6F13CHO, and C3F7CHO.

    Science.gov (United States)

    Solignac, G; Mellouki, A; Le Bras, G; Barnes, I; Benter, Th

    2006-04-06

    The Cl atom initiated oxidation of C(6)F(13)CH(2)OH, C(6)F(13)CHO, and C(3)F(7)CHO was investigated at 298 K and 1000 mbar pressure of air in a photoreactor using in situ Fourier transform infrared (FTIR) analysis. The rate coefficient for the reaction Cl + C(6)F(13)CH(2)OH (reaction 2) was measured using a relative method: k(2) = (6.5 +/- 0.8) x 10(-13) cm(3) molecule(-1) s(-1). C(6)F(13)CHO was detected as the major primary product, while CO and CF(2)O were found to be the major secondary products. A fitting procedure applied to the concentration-time profiles of C(6)F(13)CHO provided a production yield of (1.0 +/- 0.2) for this aldehyde in reaction 2, and the rate coefficient for the reaction Cl + C(6)F(13)CHO (reaction 4) was k(4) = (2.8 +/- 0.7) x 10(-12) cm(3) molecule(-1) s(-1). A high CO yield observed in the oxidation of C(6)F(13)CH(2)OH, (52 +/- 1)%, is attributed to the Cl atom initiated oxidation of C(6)F(13)CHO. High CO yields, (61 +/- 2)% and (85 +/- 5)%, were also measured in the Cl atom initiated oxidation of C(3)F(7)CHO in air and nitrogen, respectively. These high CO yields suggest the occurrence of a decomposition reaction of the perfluoroacyl, C(6)F(13)CO, and C(3)F(7)CO radicals to form CO which will compete with the combination reaction of these radicals with oxygen to form perfluoroacyl peroxy radicals in the presence of air. The latter radicals C(n)F(2)(n)(+1)CO(O)(2) (n = 6-12), through their reaction with HO(2) radicals, are currently considered as a possible source of persistent perfluorocarboxylic acids which have been detected in the environment. The consequences of the present results would be a reduction of the strength of this potential source of carboxylic acids in the atmosphere.

  2. An introduction to vectors, vector operators and vector analysis

    CERN Document Server

    Joag, Pramod S

    2016-01-01

    Ideal for undergraduate and graduate students of science and engineering, this book covers fundamental concepts of vectors and their applications in a single volume. The first unit deals with basic formulation, both conceptual and theoretical. It discusses applications of algebraic operations, Levi-Civita notation, and curvilinear coordinate systems like spherical polar and parabolic systems and structures, and analytical geometry of curves and surfaces. The second unit delves into the algebra of operators and their types and also explains the equivalence between the algebra of vector operators and the algebra of matrices. Formulation of eigen vectors and eigen values of a linear vector operator are elaborated using vector algebra. The third unit deals with vector analysis, discussing vector valued functions of a scalar variable and functions of vector argument (both scalar valued and vector valued), thus covering both the scalar vector fields and vector integration.

  3. The products of the thermal decomposition of CH{sub 3}CHO

    Energy Technology Data Exchange (ETDEWEB)

    Vasiliou, AnGayle [Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215 (United States); National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, Colorado 80401 (United States); Piech, Krzysztof M.; Barney Ellison, G. [Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215 (United States); Zhang Xu [Jet Propulsion Laboratory, California Institute of Technology, 4800 Oak Grove Drive, Pasadena, California 91109-8099 (United States); Nimlos, Mark R. [National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, Colorado 80401 (United States); Ahmed, Musahid; Golan, Amir; Kostko, Oleg [Chemical Sciences Division, Lawrence Berkeley National Laboratory, MS 6R-2100, Berkeley, California 94720 (United States); Osborn, David L. [Combustion Research Facility, Sandia National Laboratories, P.O. Box 969, MS 9055, Livermore, California 94551-0969 (United States); Daily, John W. [Center for Combustion and Environmental Research, Department of Mechanical Engineering, University of Colorado at Boulder, Boulder, Colorado 80309-0427 (United States); Stanton, John F. [Institute for Theoretical Chemistry, Department of Chemistry, University of Texas, Austin, Texas 78712 (United States)

    2011-07-07

    We have used a heated 2 cm x 1 mm SiC microtubular ({mu}tubular) reactor to decompose acetaldehyde: CH{sub 3}CHO +{Delta}{yields} products. Thermal decomposition is followed at pressures of 75-150 Torr and at temperatures up to 1675 K, conditions that correspond to residence times of roughly 50-100 {mu}s in the {mu}tubular reactor. The acetaldehyde decomposition products are identified by two independent techniques: vacuum ultraviolet photoionization mass spectroscopy (PIMS) and infrared (IR) absorption spectroscopy after isolation in a cryogenic matrix. Besides CH{sub 3}CHO, we have studied three isotopologues, CH{sub 3}CDO, CD{sub 3}CHO, and CD{sub 3}CDO. We have identified the thermal decomposition products CH{sub 3} (PIMS), CO (IR, PIMS), H (PIMS), H{sub 2} (PIMS), CH{sub 2}CO (IR, PIMS), CH{sub 2}=CHOH (IR, PIMS), H{sub 2}O (IR, PIMS), and HC{identical_to}CH (IR, PIMS). Plausible evidence has been found to support the idea that there are at least three different thermal decomposition pathways for CH{sub 3}CHO; namely, radical decomposition: CH{sub 3}CHO +{Delta}{yields} CH{sub 3}+[HCO]{yields} CH{sub 3}+ H + CO; elimination: CH{sub 3}CHO +{Delta}{yields} H{sub 2}+ CH{sub 2}=C=O; isomerization/elimination: CH{sub 3}CHO +{Delta}{yields}[CH{sub 2}=CH-OH]{yields} HC{identical_to}CH + H{sub 2}O. An interesting result is that both PIMS and IR spectroscopy show compelling evidence for the participation of vinylidene, CH{sub 2}=C:, as an intermediate in the decomposition of vinyl alcohol: CH{sub 2}=CH-OH +{Delta}{yields}[CH{sub 2}=C:]+ H{sub 2}O {yields} HC{identical_to}CH + H{sub 2}O.

  4. Vector velocimeter

    DEFF Research Database (Denmark)

    2012-01-01

    for generation of a reference beam, a detector system comprising a first detector arrangement arranged in such a way that the signal beam and the reference beam are incident upon the first detector arrangement with the reference beam propagating at an angle relative to a signal beam, and wherein the first......The present invention relates to a compact, reliable and low-cost vector velocimeter for example for determining velocities of particles suspended in a gas or fluid flow, or for determining velocity, displacement, rotation, or vibration of a solid surface, the vector velocimeter comprising a laser...... assembly for emission of a measurement beam for illumination of an object in a measurement volume with coherent light whereby a signal beam emanating from the object in the measurement volume is formed in response to illumination of the object by the measurement beam, a reference beam generator...

  5. Comparison of internal ribosome entry site (IRES and Furin-2A (F2A for monoclonal antibody expression level and quality in CHO cells.

    Directory of Open Access Journals (Sweden)

    Steven C L Ho

    Full Text Available Four versions of tricistronic vectors expressing IgG1 light chain (LC, IgG1 heavy chain (HC, and dihydrofolate reductase (DHFR in one transcript were designed to compare internal ribosome entry site (IRES and furin-2A (F2A for their influence on monoclonal antibody (mAb expression level and quality in CHO DG44 cells. LC and HC genes are arranged as either the first or the second cistron. When using mAb quantification methods based on the detection antibodies against HC Fc region, F2A-mediated tricistronic vectors appeared to express mAb at higher levels than the IRES-mediated tricistronic vectors in both transient and stable transfections. Further analysis revealed that more than 40% of products detected in stably transfected pools generated using the two F2A-mediated tricistronic vectors were aggregates. LC and HC from the F2A stably transfected pools were not properly processed, giving rise to LC+F2A+HC or HC+F2A+LC fusion proteins, LC and HC polypeptides with F2A remnants, and incorrectly cleaved signal peptides. Both IRES-mediated tricistronic vectors express mAb with correct sizes and signal peptide cleavage. Arrangement of LC as the first cistron in the IRES-mediated tricistronic vectors exhibits increased mAb expression level, better growth, and minimized product aggregation, while arrangement of HC as first cistron results in low expression, slower growth, and high aggregation. The results obtained will be beneficial for designing vectors that enhance mAb expression level and quality in mammalian cells.

  6. A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1.

    Science.gov (United States)

    Gonçalves, Cristine; Gross, Fabian; Guégan, Philippe; Cheradame, Hervé; Midou, Patrick

    2014-11-01

    Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 × 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 μg/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000™, and gene expression is higher than observed with FreeStyle™ and JetPEI®. Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Effect of fat and CHO meals on intermittent exercise in soccer players.

    Science.gov (United States)

    Hulton, A T; Edwards, J P; Gregson, W; Maclaren, D; Doran, D A

    2013-02-01

    Pre-exercise meals containing carbohydrates (CHO) are recommended to athletes, although there is evidence to suggest that a high fat meal prior to exercise increases utilisation of fats yet may not adversely affect performance. This study investigated the effect of a high fat and high CHO pre-exercise meal prior to high intensity intermittent exercise. Ten male recreational soccer players performed a soccer specific protocol followed by a 1 km time trial 3 ½ h after ingesting one of 2 test meals, high fat meal (HFM) or a high CHO meal (HCM). Blood glucose, fatty acids (FA), glycerol, β-hydroxybutyrate, lactate and insulin were assessed prior to the meal, pre-exercise, half-time, and post-exercise, whilst rates of CHO and fat oxidation were determined at 4 time points during the exercise as well as heart rate (HR) and rating of perceived exertion (RPE). Significant increases in FA, glycerol, β-hydroxybutyrate and fat oxidation after the HFM were observed, while CHO oxidation was significantly higher following the HCM (Psoccer simulated exercise has an impact on metabolism, but not on the subsequent performance as determined in the present study. © Georg Thieme Verlag KG Stuttgart · New York.

  8. Enhancement of Human Prolactin Synthesis by Sodium Butyrate Addition to Serum-Free CHO Cell Culture

    Directory of Open Access Journals (Sweden)

    Herbert Rodrigues Goulart

    2010-01-01

    Full Text Available Sodium butyrate (NaBu has been used as a productivity enhancer for the synthesis of recombinant proteins in Chinese hamster ovary (CHO cells. Thus, the influence of NaBu on the production of recombinant human prolactin (hPRL from CHO cells was investigated for the first time. CHO cell cultures were submitted to a treatment with different concentrations of NaBu (0.25 to 4 mM. Quantitative and qualitative analyses by reverse-phase high-performance liquid chromatography (RP-HPLC and Western blot or SDS-PAGE, carried out directly on CHO-conditioned medium, showed that the highest hPRL expression was obtained with 1 mM NaBu. In vitro biological assays based on noble rat lymphoma (Nb2 and mouse pro-B lymphoma (Ba/F3-LLP cells were carried out on purified hPRL. Its bioactivity in the presence of NaBu was not apparently different from that of the First International Reference Reagent of recombinant hPRL (WHO 97/714. Our results show that NaBu increased the synthesis of recombinant hPRL in CHO cells, apparently without compromising either its structure or function.

  9. Into the unknown: expression profiling without genome sequence information in CHO by next generation sequencing.

    Science.gov (United States)

    Birzele, Fabian; Schaub, Jochen; Rust, Werner; Clemens, Christoph; Baum, Patrick; Kaufmann, Hitto; Weith, Andreas; Schulz, Torsten W; Hildebrandt, Tobias

    2010-07-01

    The arrival of next-generation sequencing (NGS) technologies has led to novel opportunities for expression profiling and genome analysis by utilizing vast amounts of short read sequence data. Here, we demonstrate that expression profiling in organisms lacking any genome or transcriptome sequence information is feasible by combining Illumina's mRNA-seq technology with a novel bioinformatics pipeline that integrates assembled and annotated Chinese hamster ovary (CHO) sequences with information derived from related organisms. We applied this pipeline to the analysis of CHO cells which were chosen as a model system owing to its relevance in the production of therapeutic proteins. Specifically, we analysed CHO cells undergoing butyrate treatment which is known to affect cell cycle regulation and to increase the specific productivity of recombinant proteins. By this means, we identified sequences for >13,000 CHO genes which added sequence information of approximately 5000 novel genes to the CHO model. More than 6000 transcript sequences are predicted to be complete, as they covered >95% of the corresponding mouse orthologs. Detailed analysis of selected biological functions such as DNA replication and cell cycle control, demonstrated the potential of NGS expression profiling in organisms without extended genome sequence to improve both data quantity and quality.

  10. Enhanced transient recombinant protein production in CHO cells through the co-transfection of the product gene with Bcl-xL

    Science.gov (United States)

    Zustiak, Matthew P.; Jose, Lisa; Xie, Yueqing; Zhu, Jianwei; Betenbaugh, Micheal J.

    2014-01-01

    Transient gene expression is gaining popularity as a method to rapidly produce recombinant proteins in mammalian cells. Although significant improvements have been made, in terms of expression, more improvements are needed to compete with the yields achievable in stable gene expression. Much progress has come from optimization of transfection media and parameters, as well as altering culturing conditions to enhance productivity. Recent studies have included using cell lines engineered for apoptosis resistance through the constitutive expression of an anti-apoptotic protein, Bcl-xL. In this study we examine an alternative method of using the benefits of anti-apoptotic gene expression to enhance the transient expression of biotherapeutics, namely, through the co-transfection of bcl-xL and the product-coding gene. CHO-S cells were co-transfected with the product-coding gene and a vector containing Bcl-xL using polyethylenimine. Cells co-transfected with Bcl-xL showed reduced levels of apoptosis, increased specific productivity, and an overall increase in product yield of approximately 100%. Similar results were produced by employing another anti-apoptotic protein, Bcl-2 delta in CHO cells, or through the co-transfection with bcl-xL using HEK-293E cells. This work provides an alternative method for increasing yields of therapeutic proteins in TGE applications without generating a prior stable cell line and subsequent screening which are both time and resource consuming. PMID:24604826

  11. Increased recombinant protein production owing to expanded opportunities for vector integration in high chromosome number Chinese hamster ovary cells.

    Science.gov (United States)

    Yamano, Noriko; Takahashi, Mai; Ali Haghparast, Seyed Mohammad; Onitsuka, Masayoshi; Kumamoto, Toshitaka; Frank, Jana; Omasa, Takeshi

    2016-08-01

    Chromosomal instability is a characteristic of Chinese hamster ovary (CHO) cells. Cultures of these cells gradually develop heterogeneity even if established from a single cell clone. We isolated cells containing different numbers of chromosomes from a CHO-DG44-based human granulocyte-macrophage colony stimulating factor (hGM-CSF)-producing cell line and found that high chromosome number cells showed higher hGM-CSF productivity. Therefore, we focused on the relationship between chromosome aneuploidy of CHO cells and high recombinant protein-producing cell lines. Distribution and stability of chromosomes were examined in CHO-DG44 cells, and two cell lines expressing different numbers of chromosomes were isolated from the original CHO-DG44 cell line to investigate the effect of aneuploid cells on recombinant protein production. Both cell lines were stably transfected with a vector that expresses immunoglobulin G3 (IgG3), and specific antibody production rates were compared. Cells containing more than 30 chromosomes had higher specific antibody production rates than those with normal chromosome number. Single cell analysis of enhanced green fluorescent protein (Egfp)-gene transfected cells revealed that increased GFP expression was relative to the number of gene integration sites rather than the difference in chromosome numbers or vector locations. Our results suggest that CHO cells with high numbers of chromosomes contain more sites for vector integration, a characteristic that could be advantageous in biopharmaceutical production. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. The gas phase tropospheric removal of fluoroaldehydes (CxF2x+1CHO, x = 3, 4, 6).

    Science.gov (United States)

    Solignac, G; Mellouki, A; Le Bras, G; Yujing, Mu; Sidebottom, H

    2007-08-21

    The rate coefficient of the OH reaction with the perfluoroaldehydes C(3)F(7)CHO and C(4)F(9)CHO have been determined in the temperature range 252-373 K using the pulsed laser photolysis-laser induced fluorescence (PLP-LIF) method: k(C(3)F(7)CHO+OH) = (2.0 +/- 0.6) x 10(-12) exp[-(369 +/- 90)/T] and k(C(4)F(9)CHO+OH) = (2.0 +/- 0.5) x 10(-12) exp[-(356 +/- 70)/T] cm(3) molecule(-1) s(-1), corresponding to (5.8 +/- 0.6) x 10(-13) and (6.1 +/- 0.5) x 10(-13) cm(3) molecule(-1) s(-1), respectively, at 298 K. The UV absorption cross sections of these two aldehydes and CF(3)(CF(2))(5)CH(2)CHO have been measured over the range 230-390 nm at 298 K and also at 328 K for CF(3)(CF(2))(5)CH(2)CHO. The obtained results for C(3)F(7)CHO and C(4)F(9)CHO are in good agreement with two recent determinations but the maximum value of the absorption cross section for CF(3)(CF(2))(5)CH(2)CHO is over a factor of two lower than the single one recently published. The photolysis rates of C(3)F(7)CHO, C(4)F(9)CHO and CF(3)(CF(2))(5)CHO have been measured under sunlight conditions in the EUPHORE simulation chamber in Valencia (Spain) at the beginning of June. The photolysis rates were, respectively, J(obs) = (1.3 +/- 0.6) x 10(-5), (1.9 +/- 0.8) x 10(-5) and (0.6 +/- 0.3) x 10(-5) s(-1). From the J(obs) measurements and calculated photolysis rate J(calc), assuming a quantum yield of unity across the atmospheric range of absorption of the aldehydes, quantum yields J(obs)/J(calc) = (0.023 +/- 0.012), (0.029 +/- 0.015) and (0.046 +/- 0.028) were derived for the photodissociation of C(3)F(7)CHO, C(4)F(9)CHO and CF(3)(CF(2))(5)CHO, respectively. The atmospheric implication of the data obtained in this work is discussed. The main conclusion is that the major atmospheric removal pathway for fluoroaldehydes will be photolysis, which under low NO(x) conditions, may be a source of fluorinated carboxylic acids in the troposphere.

  13. The Use of Transcription Terminators to Generate Transgenic Lines of Chinese Hamster Ovary Cells (CHO) with Stable and High Level of Reporter Gene Expression.

    Science.gov (United States)

    Gasanov, N B; Toshchakov, S V; Georgiev, P G; Maksimenko, O G

    2015-01-01

    Mammalian cell lines are widely used to produce recombinant proteins. Stable transgenic cell lines usually contain many insertions of the expression vector in one genomic region. Transcription through transgene can be one of the reasons for target gene repression after prolonged cultivation of cell lines. In the present work, we used the known transcription terminators from the SV40 virus, as well as the human β- and γ-globin genes, to prevent transcription through transgene. The transcription terminators were shown to increase and stabilize the expression of the EGFP reporter gene in transgenic lines of Chinese hamster ovary (CHO) cells. Hence, transcription terminators can be used to create stable mammalian cells with a high and stable level of recombinant protein production.

  14. Gas phase UV and IR absorption spectra of CxF2x+1CHO (x=1-4)

    DEFF Research Database (Denmark)

    Hashikawa, Y; Kawasaki, M; Waterland, RL

    2004-01-01

    The UV and IR spectra of CxF2x+1 CHO (x = 1-4) were investigated using computational and experimental techniques. CxF2x+1CHO (x = 1-4) have broad UV absorption features centered at 300-310 nm. The maximum absorption cross-section increases significantly and shifts slightly to the red with increas...

  15. Multi-omic profiling of EPO-producing CHO cell panel reveals metabolic adaptation to heterologous protein production

    DEFF Research Database (Denmark)

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael

    The Chinese hamster ovary (CHO) cell line is the predominant mammalian cell factory for production of therapeutic glycoproteins. In this work, we aimed to study bottlenecks in the secretory pathway associated with the production of human erythropoietin (EPO) in CHO cells. In connection to this, we...

  16. Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Beuchert Kallehauge, Thomas; Pedersen, Lasse Ebdrup

    2015-01-01

    gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following...

  17. The GalNAc-type O-Glycoproteome of CHO Cells Characterized by the SimpleCell Strategy

    DEFF Research Database (Denmark)

    Zhang, Yang; Halim, Adnan; Narimatsu, Yoshiki

    2014-01-01

    The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its “human-like” glycosylation features. CHO is used for production of several O-glycoprotein therapeutics including erythropoietin, coagulation factors, ...

  18. The GalNAc-type O-Glycoproteome of CHO cells characterized by the SimpleCell strategy.

    Science.gov (United States)

    Yang, Zhang; Halim, Adnan; Narimatsu, Yoshiki; Jitendra Joshi, Hiren; Steentoft, Catharina; Schjoldager, Katrine Ter-Borch Gram; Alder Schulz, Morten; Sealover, Natalie R; Kayser, Kevin J; Paul Bennett, Eric; Levery, Steven B; Vakhrushev, Sergey Y; Clausen, Henrik

    2014-12-01

    The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its "human-like" glycosylation features. CHO is used for production of several O-glycoprotein therapeutics including erythropoietin, coagulation factors, and chimeric receptor IgG1-Fc-fusion proteins, however, some O-glycoproteins are not produced efficiently in CHO. We have previously shown that the capacity for O-glycosylation of proteins can be one limiting parameter for production of active proteins in CHO. Although the capacity of CHO for biosynthesis of glycan structures (glycostructures) on glycoproteins are well established, our knowledge of the capacity of CHO cells for attaching GalNAc-type O-glycans to proteins (glycosites) is minimal. This type of O-glycosylation is one of the most abundant forms of glycosylation, and it is differentially regulated in cells by expression of a subset of homologous polypeptide GalNAc-transferases. Here, we have genetically engineered CHO cells to produce homogeneous truncated O-glycans, so-called SimpleCells, which enabled lectin enrichment of O-glycoproteins and characterization of the O-glycoproteome. We identified 738 O-glycoproteins (1548 O-glycosites) in cell lysates and secretomes providing the first comprehensive insight into the O-glycosylation capacity of CHO (http://glycomics.ku.dk/o-glycoproteome_db/). © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Improving the representation of modal choice into bottom-up optimization energy system models - The MoCho-TIMES model

    DEFF Research Database (Denmark)

    Tattini, Jacopo; Ramea, Kalai; Gargiulo, Maurizio

    2018-01-01

    This study presents MoCho-TIMES, an original methodology for incorporating modal choice into energy-economy-environment-engineering (E4) system models. MoCho-TIMES addresses the scarce ability of E4 models to realistically depict behaviour in transport and allows for modal shift towards transit a...

  20. CHO On A Detox: Removing By-Product Formation Through Cell Engineering

    DEFF Research Database (Denmark)

    Pereira, Sara; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    Chinese Hamster Ovary (CHO) cells are the preferred hosts for the production of therapeutic glycoproteins. However, there is a need for improvement of the bioprocesses towards increased cell growth and higher productivities without compromising the product quality. Efforts to obtain tailor-made p...

  1. Network reconstruction of the mouse secretory pathway applied on CHO cell transcriptome data

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kaas, Christian Schrøder; Brandl, Julian

    2017-01-01

    to study protein secretion through graphical visualizations of omics data. We have demonstrated the use of these methods to identify potential new and known targets for engineering improved growth and IgG production, as well as the general observation that CHO cells seem to have less strict transcriptional...

  2. Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells

    DEFF Research Database (Denmark)

    Grav, Lise Marie; Julie la Cour Karottki, Karen; Lee, Jae Seong

    2017-01-01

    and yields. In this chapter, we present our protocol on how to use the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knockout engineering target genes in CHO cells. As an example, we refer to the glutamine synthetase (GS...

  3. C-terminal KDEL-modified cystatin C is retained in transfected CHO cells

    DEFF Research Database (Denmark)

    Johansen, Teit Eliot; Vogel, Charlotte Katrine; Schwartz, Thue W.

    1990-01-01

    The significance of a C-terminal tetrapeptide, Lys-Asp-Glu-Leu (KDEL), as a retention signal for the endoplasmatic reticulum was studied using cystatin C, a general thiol protease inhibitor, as the reporter protein. Clones of CHO cells were analyzed after stable transfection with eukaryotic...

  4. A proteomic study of cMyc improvement of CHO culture

    Directory of Open Access Journals (Sweden)

    Dunn Michael J

    2010-03-01

    Full Text Available Abstract Background The biopharmaceutical industry requires cell lines to have an optimal proliferation rate and a high integral viable cell number resulting in a maximum volumetric recombinant protein product titre. Nutrient feeding has been shown to boost cell number and productivity in fed-batch culture, but cell line engineering is another route one may take to increase these parameters in the bioreactor. The use of CHO-K1 cells with a c-myc plasmid allowing for over-expressing c-Myc (designated cMycCHO gives a higher integral viable cell number. In this study the differential protein expression in cMycCHO is investigated using two-dimensional gel electrophoresis (2-DE followed by image analysis to determine the extent of the effect c-Myc has on the cell and the proteins involved to give the new phenotype. Results Over 100 proteins that were differentially expressed in cMycCHO cells were detected with high statistical confidence, of which 41 were subsequently identified by tandem mass spectrometry (LC-MS/MS. Further analysis revealed proteins involved in a variety of pathways. Some examples of changes in protein expression include: an increase in nucleolin, involved in proliferation and known to aid in stabilising anti-apoptotic protein mRNA levels, the cytoskeleton and mitochondrial morphology (vimentin, protein biosysnthesis (eIF6 and energy metabolism (ATP synthetase, and a decreased regulation of all proteins, indentified, involved in matrix and cell to cell adhesion. Conclusion These results indicate several proteins involved in proliferation and adhesion that could be useful for future approaches to improve proliferation and decrease adhesion of CHO cell lines which are difficult to adapt to suspension culture.

  5. Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Grunnet, M; Angelo, K

    2002-01-01

    and oocytes. To address this problem, we have constructed a plasmid vector, pXOOM, that can function as a template for expression in both oocytes and mammalian cells. By including all the necessary RNA stability elements for oocyte expression in a standard mammalian expression vector, we have obtained a dual-function...... vector capable of supporting protein production in both Xenopus oocytes and CHO-K1 cells at an expression level equivalent to the levels obtained with vectors optimized for either oocyte or mammalian expression. Our functional studies have been performed with hERGI, KCNQ4, and Kv1.3 potassium channels....... will often engage both oocytes and mammalian cells. Efficient expression of a protein in both systems have thus far only been possible by subcloning the cDNA into two different vectors because several different molecular requirements should be fulfilled to obtain a high protein level in both mammalian cells...

  6. Modeling shear-induced CHO cell damage in a rotary positive displacement pump.

    Science.gov (United States)

    Kamaraju, Hari; Wetzel, Kenneth; Kelly, William J

    2010-01-01

    Rotary lobe pumps are commonly used in the biotechnology industry for a variety of purposes. Shear damage to animal cells within the rotary lobe pump can adversely affect the product yield or purity during, for example, cell concentration via cross-flow filtration. In this research, CHO cells grown in 20-L bioreactors were fed to a rotary lobe pump in both single pass and recycle experiments were conducted at different RPMs and "slip" conditions. The results indicate that the slip flow rate more severely impacts the viability of the CHO cells than the pump RPM. A novel mathematical modeling approach is presented that predicts shear rates in all of the positive displacement pump's slip regions, and then predicts cell death vs. operating conditions. This model accounts for the complex flow situation that results from changes to RPM, backpressure and pump geometry (i.e., clearances). Copyright © 2010 American Institute of Chemical Engineers (AIChE).

  7. Baicalein reduces oxidative stress in CHO cell cultures and improves recombinant antibody productivity

    DEFF Research Database (Denmark)

    Kwang Ha, Tae; Hansen, Anders Holmgaard; Kol, Stefan

    2017-01-01

    Oxidative stress that naturally accumulates in the endoplasmic reticulum (ER) as a result of mitochondrial energy metabolism and protein synthesis can disturb the ER function. Because ER has a responsibility on the protein synthesis and quality control of the secreted proteins, ER homeostasis has...... to be well maintained. When H2O2, an oxidative stress inducer, was added to recombinant Chinese hamster ovary (rCHO) cell cultures, it reduced cell growth, monoclonal antibody (mAb) production, and galactosylated form of mAb in a dose-dependent manner. To find an effective antioxidant for rCHO cell cultures....... Addition of baicalein significantly reduced the expression level of BiP and CHOP along with reduced reactive oxygen species level, suggesting oxidative stress accumulated in the cells can be relieved using baicalein. As a result, addition of baicalein in batch cultures resulted in 1.7 - 1.8-fold increase...

  8. Sequencing the CHO DXB11 genome reveals regional variations in genomic stability and haploidy

    DEFF Research Database (Denmark)

    Kaas, Christian Schrøder; Kristensen, Claus; Betenbaugh, Michael J.

    2015-01-01

    depths of 0x, 16x, 33x and 49x coverage corresponding to a copy number in the genome of 0, 1, 2 and 3 copies. This indicate that 17% of the genes are haploid revealing a large number of genes which can be knocked out with relative ease. This tendency of haploidy was furthermore shown to be present...... in eight additional analyzed CHO genomes (15-20% haploidy) but not in the genome of the Chinese hamster. The dhfr gene is confirmed to be haploid in CHO DXB11; transcriptionally active and the remaining allele contains a G410C point mutation causing a Thr137Arg missense mutation. We find similar to 2...

  9. Quantitative intracellular flux modeling and applications in biotherapeutic development and production using CHO cell cultures.

    Science.gov (United States)

    Huang, Zhuangrong; Lee, Dong-Yup; Yoon, Seongkyu

    2017-12-01

    Chinese hamster ovary (CHO) cells have been widely used for producing many recombinant therapeutic proteins. Constraint-based modeling, such as flux balance analysis (FBA) and metabolic flux analysis (MFA), has been developing rapidly for the quantification of intracellular metabolic flux distribution at a systematic level. Such methods would produce detailed maps of flows through metabolic networks, which contribute significantly to better understanding of metabolism in cells. Although these approaches have been extensively established in microbial systems, their application to mammalian cells is sparse. This review brings together the recent development of constraint-based models and their applications in CHO cells. The further development of constraint-based modeling approaches driven by multi-omics datasets is discussed, and a framework of potential modeling application in cell culture engineering is proposed. Improved cell culture system understanding will enable robust developments in cell line and bioprocess engineering thus accelerating consistent process quality control in biopharmaceutical manufacturing. © 2017 Wiley Periodicals, Inc.

  10. Comprehensible Input as Sociocognitive Alignment: A Response to Cho and Krashen (2016)

    OpenAIRE

    Sugiharto, Setiono

    2016-01-01

    A plethora of studies on how language is acquired through comprehensible inputs has generated valuable insights into language acquisition theory. Many of these studies have confirmed that humans acquire language in one way – through reading and listening. In particular, a recent study by Cho and Krashen (2016) published in this journal further confirms that the exposure to input (i.e. in the form of pleasure reading) is beneficial for attaining advanced level of language development both in a...

  11. Comprehensible Input as Sociocognitive Alignment: a Response to Cho and Krashen (2016)

    OpenAIRE

    Sugiharto, Setiono

    2016-01-01

    A plethora of studies on how language is acquired through comprehensible inputs has generated valuable insights into language acquisition theory. Many of these studies have confirmed that humans acquire language in one way – through reading and listening. In particular, a recent study by Cho and Krashen (2016) published in this journal further confirms that the exposure to input (i.e. in the form of pleasure reading) is beneficial for attaining advanced level of language development both in a...

  12. The Golgi CMP-sialic acid transporter: A new CHO mutant provides functional insights.

    Science.gov (United States)

    Lim, Sing Fee; Lee, May May; Zhang, Peiqing; Song, Zhiwei

    2008-11-01

    A CHO mutant line, MAR-11, was isolated using a cytotoxic lectin, Maackia amurensis agglutinin (MAA). This mutant has decreased levels of cell surface sialic acid relative to both wild-type CHO-K1 and Lec2 mutant CHO cells. The CMP-sialic acid transporter (CMP-SAT) gene in the MAR-11 mutant cell has a C-T mutation that results in a premature stop codon. As a result, MAR-11 cells express a truncated version of CMP-SAT which contains only 100 amino acids rather than the normal CMP-SAT which contains 336 amino acids. Biochemical analyses indicate that recombinant interferon-gamma (IFN-gamma) produced by the mutant cells lack sialic acid. Using MAR-11 as host cells, an EPO/IEF assay for the structure-function study of CMP-SAT was developed. This assay seems more sensitive than previous assays that were used to analyze sialylation in Lec2 cells. Cotransfection of constructs that express CMP-SAT into MAR-11 cells completely converted the recombinant EPO to a sialylation pattern that is similar to the EPO produced by the wild-type CHO cells. Using this assay, we showed that CMP-SAT lacking C-terminal 18 amino acids from the cytosolic tail was able to allow high levels of EPO sialylation. Substitution of the Gly residues with Ile in three different transmembrane domains of CMP-SAT resulted in dramatic decreases in transporter's activity. The CMP-SAT only lost partial activity if the same Gly residues were substituted with Ala, suggesting that the lack of side chain in Gly residues in the transmembrane domains is essential for transport activity.

  13. Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

    Directory of Open Access Journals (Sweden)

    Qi He

    Full Text Available The endocytosis of transferrin receptor (TfR has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

  14. Precision control of recombinant gene transcription for CHO cell synthetic biology.

    Science.gov (United States)

    Brown, Adam J; James, David C

    2016-01-01

    The next generation of mammalian cell factories for biopharmaceutical production will be genetically engineered to possess both generic and product-specific manufacturing capabilities that may not exist naturally. Introduction of entirely new combinations of synthetic functions (e.g. novel metabolic or stress-response pathways), and retro-engineering of existing functional cell modules will drive disruptive change in cellular manufacturing performance. However, before we can apply the core concepts underpinning synthetic biology (design, build, test) to CHO cell engineering we must first develop practical and robust enabling technologies. Fundamentally, we will require the ability to precisely control the relative stoichiometry of numerous functional components we simultaneously introduce into the host cell factory. In this review we discuss how this can be achieved by design of engineered promoters that enable concerted control of recombinant gene transcription. We describe the specific mechanisms of transcriptional regulation that affect promoter function during bioproduction processes, and detail the highly-specific promoter design criteria that are required in the context of CHO cell engineering. The relative applicability of diverse promoter development strategies are discussed, including re-engineering of natural sequences, design of synthetic transcription factor-based systems, and construction of synthetic promoters. This review highlights the potential of promoter engineering to achieve precision transcriptional control for CHO cell synthetic biology. Copyright © 2015. Published by Elsevier Inc.

  15. Silver nanoparticle induced cytotoxicity, oxidative stress, and DNA damage in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Awasthi, Kumud Kant [University of Rajasthan, Department of Zoology (India); Awasthi, Anjali; Kumar, Narender; Roy, Partha [Indian Institute of Technology Roorkee, Department of Biotechnology (India); Awasthi, Kamlendra, E-mail: kamlendra.awasthi@gmail.com [Malaviya National Institute of Technology, Department of Physics (India); John, P. J., E-mail: placheriljohn@yahoo.com [University of Rajasthan, Department of Zoology (India)

    2013-09-15

    Silver nanoparticles (Ag NPs) are being used increasingly in wound dressings, catheters, and in various household products due to their antimicrobial activity. The present study reports the toxicity evaluation of synthesized and well characterized Ag NPs using Chinese hamster ovary (CHO) cells. The UV-Vis spectroscopy reveals the formation of silver nanoparticles by exhibiting the typical surface plasmon absorption maxima at 408-410 nm. Transmission electron microscopy (TEM) reveals that the average diameter of silver nanoparticles is about 5.0 {+-} 1.0 nm and that they have spherical shape. Cell visibility and cell viability percentage show dose-dependent cellular toxicity of Ag NPs. The half maximal inhibitory concentration (IC{sub 50}) for CHO cells is 68.0 {+-} 2.65 {mu}g/ml after 24 h Ag NPs exposure. Toxicity evaluations, including cellular morphology, mitochondrial function (MTT assay), reactive oxygen species (ROS), and DNA fragmentation assay (Ladder pattern) were assessed in unexposed CHO cells (control) and the cells exposed to Ag NPs concentrations of 15, 30, and 60 {mu}g/ml for 24 h. The findings may assist in the designing of Ag NPs for various applications and provide insights into their toxicity.

  16. Absence of micronucleus formation in CHO-K1 cells cultivated in platelet lysate enriched medium.

    Science.gov (United States)

    Bernardi, Martina; Adami, Valentina; Albiero, Elena; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

    2014-03-01

    Human platelet lysate (PL) represents an effective substitute of fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) cultivation. Compared to FBS, PL favors MSC proliferation significantly shortening the population doubling time and avoiding the risks related to the use of animal derivatives. Growth factors contained in the platelets are released upon platelet disruption following freezing/thawing cycles or as we have recently described by using ultrasound. We have investigated whether the increased cell proliferation achieved by using PL could induce mitotic stress and whether the potential formation of free radicals during PL production by ultrasound could cause chromosomal instability in mammalian cells. We have applied an image analysis assisted high content screening (HCS) in vitro micronucleus assay in the Chinese Hamster Ovarian K1 (CHO-K1) rodent mammalian cell line. PL was produced by sonication; for the micronucleus assay, CHO-K1 cells were exposed to increasing concentrations of PL. Cytokinesis was blocked by cytochalasin B, nuclei were stained with bisbenzimide and images were acquired and analyzed automatically using an HCS system, both with a 20× and a 10× objective. Our results suggest that growth stimulus induced by the use of PL did not significantly increase micronucleus formation in CHO-K1 cells compared to negative control. Micronucleus testing in conjunction with HCS could represent a valid tool to evaluate the safety of ancillary materials used in the production of cell-based medicinal products. Copyright © 2013 Elsevier GmbH. All rights reserved.

  17. Application of factorial design to accelerate identification of CHO growth factor requirements.

    Science.gov (United States)

    Chun, Chung; Heineken, Katy; Szeto, Dongmei; Ryll, Thomas; Chamow, Steve; Chung, John D

    2003-01-01

    To accelerate recombinant CHO media and process development, we describe a simple approach to integrating multiple tasks associated with these processes including initial media design, serum-free adaptation, stability analysis and first generation scale-up. Factorial design techniques and normal probability chart representation of the results were first applied to identify potent parental CHO cell growth factors in a lean basal medium. These results were then applied to identify a suitable manufacturing medium from a panel of commercial and proprietary media formulations. When this approach was applied to recombinant CHO cell line, rapid adaptation of the cell line to an appropriate production medium occurred during culture expansion in the presence of the identified growth factor(s). This approach allows media component screening to be naturally integrated into the adaptation and scale-up processes since components that have little or no relative effect on cell proliferation are selected against as the "best" cultures are moved forward. The rapidity of the adaptation process allowed cell line stability studies to be initiated relatively early in the development process, thus providing preliminary stability information by the time the "outgrowing" culture could be scaled to 100-L reactors some 30 days after adaptation commenced. The application of full factorial design techniques allowed us to calculate the maximum number of interaction effects, the interpretation of which we believe can provide insights into growth factor biology.

  18. Application of PCDA/SPH/CHO/Lysine vesicles to detect pathogenic bacteria in chicken.

    Science.gov (United States)

    de Oliveira, Taíla V; Soares, Nilda de F F; de Andrade, Nélio J; Silva, Deusanilde J; Medeiros, Eber Antônio A; Badaró, Amanda T

    2015-04-01

    During the course of infection, Salmonella must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments, as lysine decarboxylation to cadaverine. The idea of Salmonella defenses responses could be employed in systems as polydiacetylene (PDA) to detect this pathogen so important to public health system. Beside that PDA is an important substance because of the unique optical property; that undergoes a colorimetric transitions by various external stimuli. Therefore 10,12-pentacosadyinoic acid (PCDA)/Sphingomyelin(SPH)/Cholesterol(CHO)/Lysine system was tested to determine the colorimetric response induced by Salmonella choleraesuis. PCDA/SPH/CHO/Lysine vesicles showed a colour change even in low S. choleraesuis concentration present in laboratory conditions and in chicken meat. Thus, this work showed a PCDA/SPH/CHO/Lysine vesicle application to simplify routine analyses in food industry, as chicken meat industry. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells.

    Science.gov (United States)

    Chung, Cheng-Yu; Wang, Qiong; Yang, Shuang; Ponce, Sean A; Kirsch, Brian J; Zhang, Hui; Betenbaugh, Michael J

    2017-12-01

    One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit altered properties for these important biomedical modalities. In this study, the disruption of two α-2,3 sialyltransferases (ST3GAL4 and ST3GAL6) from Chinese Hamster Ovary (CHO) cells was combined with protein engineering of the Fc region to generate an IgG containing 80% bigalactosylated and fucosylated (G2F) glycoforms. Expression of the same single amino acid mutant (F241A) IgG in CHO cells with a triple gene knockout of fucosyltransferase (FUT8) plus ST3GAL4 and ST3GAL6 lowered the galactosylation glycoprofile to 65% bigalactosylated G2 glycans. However, overexpression of IgGs with four amino acid substitutions recovered the G2 glycoform composition approximately 80%. Combining genome and protein engineering in CHO cells will provide a new antibody production platform that enables biotechnologists to generate glycoforms standards for specific biomedical and biotechnology applications. © 2017 Wiley Periodicals, Inc.

  20. Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

    National Research Council Canada - National Science Library

    Lee, Jae Seong; Kallehauge, Thomas Beuchert; Pedersen, Lasse Ebdrup; Kildegaard, Helene Faustrup

    2015-01-01

    .... Here we demonstrate efficient targeted gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI...

  1. Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes.

    Science.gov (United States)

    Ramirez-Gordillo, Daniel; Trujillo-Provencio, Casilda; Knight, V Bleu; Serrano, Elba E

    2011-10-01

    The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine™ 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights™ Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights™ Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel β1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology.

  2. One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment

    DEFF Research Database (Denmark)

    Grav, Lise Marie; Lee, Jae Seong; Thomsen, Signe Gerling

    2015-01-01

    The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously....... Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further....

  3. Polymer-mediated flocculation of transient CHO cultures as a simple, high throughput method to facilitate antibody discovery.

    Science.gov (United States)

    Schmitt, Matthew G; Rajendra, Yashas; Hougland, Maria D; Boyles, Jeffrey S; Barnard, Gavin C

    2017-09-01

    Most biopharmaceutical drugs, especially monoclonal antibodies (mAbs), bispecific antibodies (BsAbs) and Fc-fusion proteins, are expressed using Chinese Hamster Ovary (CHO) cell lines. CHO cells typically yield high product titers and high product quality. Unfortunately, CHO cell lines also generate high molecular weight (HMW) aggregates of the desired product during cell culture along with CHO host cell protein (HCP) and CHO DNA. These immunogenic species, co-purified during Protein A purification, must be removed in a multi-step purification process. Our colleagues have reported the use of a novel polymer-mediated flocculation step to simultaneously reduce HMW, HCP and DNA from stable CHO cell cultures prior to Protein A purification. The objective of this study was to evaluate this novel "smart polymer" (SmP) in a high throughput antibody discovery workflow using transiently transfected CHO cultures. SmP treatment of 19 different molecules from four distinct molecular categories (human mAbs, murine mAbs, BsAbs and Fabs) with 0.1% SmP and 25 mM stimulus resulted in minimal loss of monomeric protein. Treatment with SmP also demonstrated a variable, concentration-dependent removal of HMW aggregates after Protein A purification. SmP treatment also effectively reduced HCP levels at each step of mAb purification with final HCP levels being several fold lower than the untreated control. Interestingly, SmP treatment was able to significantly reduce high concentrations of artificially spiked levels of endotoxin in the cultures. In summary, adding a simple flocculation step to our existing transient CHO process reduced the downstream purification burden to remove impurities and improved final product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1393-1400, 2017. © 2017 American Institute of Chemical Engineers.

  4. A signature of 12 microRNAs is robustly associated with growth rate in a variety of CHO cell lines.

    Science.gov (United States)

    Klanert, Gerald; Jadhav, Vaibhav; Shanmukam, Vinoth; Diendorfer, Andreas; Karbiener, Michael; Scheideler, Marcel; Bort, Juan Hernández; Grillari, Johannes; Hackl, Matthias; Borth, Nicole

    2016-10-10

    As Chinese Hamster Ovary (CHO) cells are the cell line of choice for the production of human-like recombinant proteins, there is interest in genetic optimization of host cell lines to overcome certain limitations in their growth rate and protein secretion. At the same time, a detailed understanding of these processes could be used to advantage by identification of marker transcripts that characterize states of performance. In this context, microRNAs (miRNAs) that exhibit a robust correlation to the growth rate of CHO cells were determined by analyzing miRNA expression profiles in a comprehensive collection of 46 samples including CHO-K1, CHO-S and CHO-DUKXB11, which were adapted to various culture conditions, and analyzed in different growth stages using microarrays. By applying Spearman or Pearson correlation coefficient criteria of>|0.6|, miRNAs with high correlation to the overall growth, or growth rates observed in exponential, serum-free, and serum-free exponential phase were identified. An overlap of twelve miRNAs common for all sample sets was revealed, with nine positively and three negatively correlating miRNAs. The here identified panel of miRNAs can help to understand growth regulation in CHO cells and contains putative engineering targets as well as biomarkers for cell lines with advantageous growth characteristics. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Prediction and cloning linear Tcell epitopes of P14-3-3 antigen into pEGFP–N1 as a DNA vaccine model to induse immuno response against hydatidosis and it\\'s expression in CHO cell line

    Directory of Open Access Journals (Sweden)

    R mesri

    2015-11-01

    Full Text Available ABSTRACT Background & purpose: Hydatidosis is a zoonotic disease that caused by infection with the larvae of Echinococcus granulosus. Different antigens produced in larval stage of this parasite that recombinant vaccine base these antigens created significant immunity in infected animals. One of the important antigens is p14-3-3 that it's recombinant antigen created considerable immunity in mouse models. In this study according to the high immunity of antigen epitopes region the coding sequence of T-cell epitopes of P14-3-3 was cloned into pEGFP-N1vector in order to produce an effective DNA vaccine model to stimulate high level of Th1 immune response.   Material and method: In this study bioinformatics tools were used to prediction of linear T-Cell epitopes of Echinococcus granulosus P14-3-3 &zeta antigen. The nucleotide coding sequence of these epitopes was synthesized by PCR. the ampliqon was digested with XhoI restriction enzyme and cloned into pEGFP–N1 vector That has been purificated by modified sambrook method with CaCl2 and PEG6000..Positive colony was selected by direct colony PCR and confirmed by the sequencing.and evaluation of it's expression in Eukaryotic cells was done by transformed to CHO cell line with electroporation. Results: Linear T-cell epitopes of Echinococcus granulosus P14-3-3 after prediction,synthesis and amplification wae successfully cloned into pEGFP-N1 vector that purificated by new method with maximum vector and minimum RNA concentration.The expression of new constract in CHO cell line as a eukaryotic cells achivment by fluorescent microscope and will be used as a DNA vaccine model to evaluation immuno response in mouse models.   Discussion: Successfully cloning of The linear T-cell epitppes coding sequence of Echinococcus granulosus P14-3-3&zeta antigen into pEGFP-N1 verificated by sequencing and fluorscent microscope images demonstrated expression of recombinant protein in CHO cell line

  6. VectorBase

    Data.gov (United States)

    U.S. Department of Health & Human Services — VectorBase is a Bioinformatics Resource Center for invertebrate vectors. It is one of four Bioinformatics Resource Centers funded by NIAID to provide web-based...

  7. Yeast PAH1-encoded phosphatidate phosphatase controls the expression of CHO1-encoded phosphatidylserine synthase for membrane phospholipid synthesis.

    Science.gov (United States)

    Han, Gil-Soo; Carman, George M

    2017-08-11

    The PAH1-encoded phosphatidate phosphatase (PAP), which catalyzes the committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae, exerts a negative regulatory effect on the level of phosphatidate used for the de novo synthesis of membrane phospholipids. This raises the question whether PAP thereby affects the expression and activity of enzymes involved in phospholipid synthesis. Here, we examined the PAP-mediated regulation of CHO1-encoded phosphatidylserine synthase (PSS), which catalyzes the committed step for the synthesis of major phospholipids via the CDP-diacylglycerol pathway. The lack of PAP in the pah1Δ mutant highly elevated PSS activity, exhibiting a growth-dependent up-regulation from the exponential to the stationary phase of growth. Immunoblot analysis showed that the elevation of PSS activity results from an increase in the level of the enzyme encoded by CHO1 Truncation analysis and site-directed mutagenesis of the CHO1 promoter indicated that Cho1 expression in the pah1Δ mutant is induced through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. The abrogation of Cho1 induction and PSS activity by a CHO1 UASINO mutation suppressed pah1Δ effects on lipid synthesis, nuclear/endoplasmic reticulum membrane morphology, and lipid droplet formation, but not on growth at elevated temperature. Loss of the DGK1-encoded diacylglycerol kinase, which converts diacylglycerol to phosphatidate, partially suppressed the pah1Δ-mediated induction of Cho1 and PSS activity. Collectively, these data showed that PAP activity controls the expression of PSS for membrane phospholipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Effect of preexercise glycemic-index meal on running when CHO-electrolyte solution is consumed during exercise.

    Science.gov (United States)

    Wong, Stephen H S; Chan, Oi Won; Chen, Ya Jun; Hu, Heng Long; Lam, Chin Wan; Chung, Pak Kwong

    2009-06-01

    This study examined the effect of consuming carbohydrate- (CHO) electrolyte solution on running performance after different-glycemic-index (GI) meals. Nine men completed 3 trials in a randomized counterbalanced order, with trials separated by at least 7 days. Two hours before the run after an overnight fast, each participant consumed a high-GI (GI = 83) or low-GI (GI = 36) CHO meal or low-energy sugar-free Jell-O (GI = 0, control). The 2 isocaloric GI meals provided 1.5 g available CHO/kg body mass. During each trial, 2 ml/kg body mass of a 6.6% CHO-electrolyte solution was provided immediately before exercise and every 2.5 km after the start of running. Each trial consisted of a 21-km performance run on a level treadmill. The participants were required to run at 70% VO2max during the first 5 km of the run. They then completed the remaining 16 km as fast as possible. There was no difference in the time to complete the 21-km run (high-GI vs. low-GI vs. control: 91.1 +/- 2.0 vs. 91.8 +/- 2.2 vs. 92.9 +/- 2.0 min, n.s.). There were no differences in total CHO and fat oxidation throughout the trials, despite differences in preexercise blood glucose, serum insulin, and serum free-fatty-acid concentrations. When a CHO-electrolyte solution is consumed during a 21-km run, the GI of the preexercise CHO meal makes no difference in running performance.

  9. Custodial vector model

    DEFF Research Database (Denmark)

    Becciolini, Diego; Franzosi, Diogo Buarque; Foadi, Roshan

    2015-01-01

    We analyze the Large Hadron Collider (LHC) phenomenology of heavy vector resonances with a $SU(2)_L\\times SU(2)_R$ spectral global symmetry. This symmetry partially protects the electroweak S-parameter from large contributions of the vector resonances. The resulting custodial vector model spectrum...

  10. Insulated Foamy Viral Vectors

    Science.gov (United States)

    Browning, Diana L.; Collins, Casey P.; Hocum, Jonah D.; Leap, David J.; Rae, Dustin T.; Trobridge, Grant D.

    2016-01-01

    Retroviral vector-mediated gene therapy is promising, but genotoxicity has limited its use in the clinic. Genotoxicity is highly dependent on the retroviral vector used, and foamy viral (FV) vectors appear relatively safe. However, internal promoters may still potentially activate nearby genes. We developed insulated FV vectors, using four previously described insulators: a version of the well-studied chicken hypersensitivity site 4 insulator (650cHS4), two synthetic CCCTC-binding factor (CTCF)-based insulators, and an insulator based on the CCAAT box-binding transcription factor/nuclear factor I (7xCTF/NF1). We directly compared these insulators for enhancer-blocking activity, effect on FV vector titer, and fidelity of transfer to both proviral long terminal repeats. The synthetic CTCF-based insulators had the strongest insulating activity, but reduced titers significantly. The 7xCTF/NF1 insulator did not reduce titers but had weak insulating activity. The 650cHS4-insulated FV vector was identified as the overall most promising vector. Uninsulated and 650cHS4-insulated FV vectors were both significantly less genotoxic than gammaretroviral vectors. Integration sites were evaluated in cord blood CD34+ cells and the 650cHS4-insulated FV vector had fewer hotspots compared with an uninsulated FV vector. These data suggest that insulated FV vectors are promising for hematopoietic stem cell gene therapy. PMID:26715244

  11. Protein A-mouse acidic mammalian chitinase-V5-His expressed in periplasmic space of Escherichia coli possesses chitinase functions comparable to CHO-expressed protein.

    Directory of Open Access Journals (Sweden)

    Akinori Kashimura

    Full Text Available Acidic mammalian chitinase (AMCase has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His6 tag (V5-His at the C-terminus (Protein A-AMCase-V5-His in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N'-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.

  12. Protein A-Mouse Acidic Mammalian Chitinase-V5-His Expressed in Periplasmic Space of Escherichia coli Possesses Chitinase Functions Comparable to CHO-Expressed Protein

    Science.gov (United States)

    Kida, Yuta; Iwabuchi, Kokoro; Matsushima, Yudai; Sakaguchi, Masayoshi; Sugahara, Yasusato; Oyama, Fumitaka

    2013-01-01

    Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)6 tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N′-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase. PMID:24244337

  13. Microprojectile Bombardment Transformation of Date Palm Using the Insecticidal Cholesterol Oxidase (ChoA) Gene.

    Science.gov (United States)

    Allam, Mai A; Saker, Mahmoud M

    2017-01-01

    The overall objective of this work is to optimize the transformation system for date palm as a first step toward production of date palm clones resistant to noxious pests. A construct harboring the cholesterol oxidase (ChoA) gene, which renders plant resistance against insect attack, is introduced into embryogenic date palm callus using the PDS-1000/He particle bombardment system. The process involves the establishment of embryogenic callus cultures as well as immature embryo-derived microcalli that are used as target tissues for shooting and optimization of transformation conditions. This chapter in addition explains molecular and histochemical assays conducted to confirm gene integration and expression.

  14. Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Jones, Meredith B., E-mail: mbauman7@jhu.edu [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Maryland Hall 221, Baltimore, MD 21218 (United States); Tomiya, Noboru, E-mail: ntomiya1@jhu.edu [Department of Biology, Johns Hopkins University, 3400 North Charles Street, Mudd Hall 104A, Baltimore, MD 21218 (United States); Betenbaugh, Michael J., E-mail: beten@jhu.edu [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Maryland Hall 221, Baltimore, MD 21218 (United States); Krag, Sharon S., E-mail: skrag@jhsph.edu [Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205 (United States)

    2010-04-23

    Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man{sub 5}GlcNAc{sub 2}-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man{sub 9}GlcNAc{sub 2}-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man{sub 5}GlcNAc{sub 2}-PP-Dol through Glc{sub 1}Man{sub 9}GlcNAc{sub 2}-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

  15. Influence of catechins on bystander responses in CHO cells induced by alpha-particle irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Law, Y.L.; Wong, T.P.W. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Yu, K.N. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong)], E-mail: peter.yu@cityu.edu.hk

    2010-04-15

    In this work, we studied alpha-particle induced and medium-mediated bystander effects in Chinese hamster ovary (CHO) cells through micronucleus (MN) assay. We showed that signal transduction from irradiated cells to bystander cells occur within a short time after irradiation. We then studied the effects of ROS (reactive oxygen species)-scavenging catechins in the medium before irradiation. We observed decreases in the percentage of bystander cells with MN formation and thus proved the protection effect of catechins on bystander cells from radiation.

  16. Approximate thermochemical tables for some C-H and C-H-O species

    Science.gov (United States)

    Bahn, G. S.

    1973-01-01

    Approximate thermochemical tables are presented for some C-H and C-H-O species and for some ionized species, supplementing the JANAF Thermochemical Tables for application to finite-chemical-kinetics calculations. The approximate tables were prepared by interpolation and extrapolation of limited available data, especially by interpolations over chemical families of species. Original estimations have been smoothed by use of a modification for the CDC-6600 computer of the Lewis Research Center PACl Program which was originally prepared for the IBM-7094 computer Summary graphs for various families show reasonably consistent curvefit values, anchored by properties of existing species in the JANAF tables.

  17. Genetically modified CHO cells for studying the genotoxicity of heterocyclic amines from cooked foods

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, L.H.; Wu, R.W.; Felton, J.S.

    1995-07-01

    We have developed metabolically competent CHO cells to evaluate the genotoxicity associated with heterocyclic amines, such as those that are present in cooked foods. Into repair-deficient UV5 cells we introduced cDNAs for expressing cytochrome P450IA2 and acetyltransferases. We then genetically reverted these transformed lines to obtain matched metabolically competent repair-deficient/proficient lines. For a high mutagenic response, we find a requirement for acetyltransferase with IQ but not with PhIP. This system allows for both quantifying mutagenesis and analyzing the mutational spectra produced by heterocyclic amines.

  18. Differential protein expression following low temperature culture of suspension CHO-K1 cells

    Directory of Open Access Journals (Sweden)

    Henry Michael

    2008-04-01

    Full Text Available Abstract Background To ensure maximal productivity of recombinant proteins (rP during production culture it is typical to encourage an initial phase of rapid cell proliferation to achieve high biomass followed by a stationary phase where cellular energies are directed towards production of rP. During many such biphasic cultures, the initial phase of rapid cell growth at 37°C is followed by a growth arrest phase induced through reduction of the culture temperature. Low temperature induced growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase, although the mechanisms regulating these phenotypes during mild hypothermia are poorly understood. Results In this study differential protein expression in suspension CHO-K1 cells was investigated following a reduction of the culture temperature from 37°C to 31°C in comparison to standard batch culture maintained at 37°C using 2D-DIGE (Fluorescence 2-D Difference Gel Electrophoresis and mass spectrometry (MS. There is only limited proteomic analysis of suspension-grown CHO cells describing a direct comparison of temperature shifted versus non-temperature shifted cultures using 2D-DIGE. This investigation has enabled the identification of temperature-dependent as well as temperature-independent proteomic changes. 201 proteins were observed as differentially expressed following temperature shift, of which 118 were up regulated. Of the 53 proteins identified by MALDI-ToF MS, 23 were specifically differentially expressed upon reduction of the culture temperature and were found related to a variety of cellular functions such as regulation of growth (HNRPC, cap-independent translation (EIF4A, apoptosis (importin-α, the cytoskeleton (vimentin and glycoprotein quality control (alpha glucosidase 2. Conclusion These results indicate the extent of the temperature response in CHO-K1 cells and suggest a number of key

  19. Macro motion vector quantization

    Science.gov (United States)

    Lee, Yoon Y.; Woods, John W.

    1995-04-01

    A new algorithm is developed for reducing the bit rate required for motion vectors. This algorithm is a generalization of block matching motion estimation in which the search region is represented as a codebook of motion vectors. The new algorithm, called macro motion vector quantization (MMVQ), generalized our earlier MVQ by coding a group of motion vectors. The codebook is a set of macro motion vectors which represent the block locations of the small neighboring blocks in the previous frame. We develop an interative design algorithm for the codebook. Our experiments show that the variances of displaced frame differences (DFDs) are reduced significantly compared to block matching algorithm (BMA) with the macroblock size.

  20. Cross-cultural adaptation of the CHO-KLAT for boys with hemophilia in rural and urban china

    Directory of Open Access Journals (Sweden)

    Wu Runhui

    2012-09-01

    Full Text Available Abstract Background Quality of life (QoL is increasingly recognized as an important outcome measure in clinical trials. The Canadian Hemophilia Outcomes-Kids Life Assessment Tool (CHO-KLAT shows promise for use in China. Objective To adapt the CHO-KLAT version 2.0 for use in clinical trials in China. Methods Forward and back translations of the CHO-KLAT2.0 were completed in 2008. Between October 2009 and June 2010, a series of 3 focus groups were held with 20 boys and 31 parents in rural and urban China to elicit additional concepts, important to their QoL, for the Chinese CHO-KLAT2.0. All of the items identified by boys and parents were reviewed by a group of experts, resulting in a Chinese version of the CHO-KLAT2.0. This version underwent a detailed cognitive debriefing process between October 2010 and June 2011. Thirteen patient-parent pairs participated in this cognitive debriefing process until a stable and clearly understood Chinese version of the CHO-KLAT2.0 was obtained. Results The initial back translation of the Chinese CHO-KLAT2.0 was slightly discrepant from the original English version on 12 items. These were all successfully adjudicated. The focus groups identified 9 new items that formed an add-on Socio-Economic Context (SEC module for China. Linguistic improvements were made after the 2nd, 5th, 7th and 13th cognitive debriefings pairs and affected a total of 18 items. The result was a 35 item CHO-KLAT2.0 and a SEC module in Simplified Chinese, both of which have good content validity. Conclusion This detailed process proved to be extremely valuable in ensuring the items were accurately interpreted by Chinese boys with hemophilia ages ≤18 years. The need for the additional SEC module highlighted the different context that currently exists in China with regard to hemophilia care as compared to many Western countries, and will be important in tracking progress within both rural and urban China over time. Changes based on the

  1. Identifying the differences in mechanisms of mycophenolic acid controlling fucose content of glycoproteins expressed in different CHO cell lines.

    Science.gov (United States)

    Zhang, An; Tsang, Valerie Liu; Markely, Lam R; Kurt, Lutfiye; Huang, Yao-Ming; Prajapati, Shashi; Kshirsagar, Rashmi

    2016-11-01

    In the biopharmaceutical industry, glycosylation is a critical quality attribute that can modulate the efficacy of a therapeutic glycoprotein. Obtaining a consistent glycoform profile is desired because molecular function can be defined by its carbohydrate structures. Specifically, the fucose content of oligosaccharides in glycoproteins is one of the most important attributes that can significantly affect antibody-dependent cellular cytotoxicity (ADCC) activity. It is therefore important to understand the fucosylation pathway and be able to control fucosylation at the desired level to match predecessor materials in late stage and biosimilar programs. Several strategies were explored in this study and mycophenolic acid (MPA) was able to finely modulate the fucose content with the least undesired side effects. However, the response was significantly different between CHO cell lines of different lineages. Further experiments were then performed for a deeper understanding of the mechanism of fucosylation in different CHO cell lines. Results indicated that changes in the intracellular nucleotide involved in fucosylation pathway after MPA treatment are the main cause of the differences in fucosylation level response in different CHO cell lines. Differences in MPA metabolism in the various CHO cell lines directly resulted in different levels of afucosylation measured in antibodies produced by the CHO cell lines. Biotechnol. Bioeng. 2016;113: 2367-2376. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. Adhesion and migration of CHO cells on micropatterned single layer graphene

    Science.gov (United States)

    Keshavan, S.; Oropesa-Nuñez, R.; Diaspro, A.; Canale, C.; Dante, S.

    2017-06-01

    Cell patterning technology on single layer graphene (SLG) is a fairly new field that can find applications in tissue engineering and biomaterial/biosensors development. Recently, we have developed a simple and effective approach for the fabrication of patterned SLG substrates by laser micromachining, and we have successfully applied it for the obtainment of geometrically ordered neural networks. Here, we exploit the same approach to investigate the generalization of the cell response to the surface cues of the fabricated substrates and, contextually, to quantify cell adhesion on the different areas of the patterns. To attain this goal, we tested Chinese hamster ovary (CHO) cells on PDL-coated micropatterned SLG substrates and quantified the adhesion by using single cell force spectroscopy (SCFS). Our results indicate higher cell adhesion on PDL-SLG, and, consequently, an initial CHO cell accumulation on the graphene areas, confirming the neuronal behaviour observed previously; interestingly, at later time point in culture, cell migration was observed towards the adjacent SLG ablated regions, which resulted more favourable for cell proliferation. Therefore, our findings indicate that the mechanism of interaction with the surface cues offered by the micropatterned substrates is strictly cell-type dependent.

  3. Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.

    Science.gov (United States)

    Zhu, Jiang; Wooh, Jong Wei; Hou, Jeff Jia Cheng; Hughes, Benjamin S; Gray, Peter P; Munro, Trent P

    2012-01-01

    Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  4. Cytotoxic effects and morphological changes of silver nanoparticles in CHO-K1 cells

    Directory of Open Access Journals (Sweden)

    Masoumeh Heshmati

    2015-11-01

    Full Text Available Background: Silver nanoparticles are of interest to be used as antimicrobial agents in medical care, wound dressings and cosmetics. Despite the fact that AgNPs are among the most commercialized nano materials owing to their specific antimicrobial properties, there is limited information about their risk assessment and possible hazards to human health and environment. Thus, this study was carried out to evaluate the cytotoxicity and morphological changes of different concentrations of two samples of commercialized silver nanoparticles (A and B in CHO-K1 cells using MTT assay. Methods: The cytotoxicity effect of silver nanoparticles AgNP-A (19.6 nm in diameter and AgNP-B (15 nm in diameter on CHO-K1 was evaluated in the range of 0.005-500 µg/ml after 24 hours of treatment using MTT assay. The morphological changes of treated cells were examined by light microscopy. Results: Based on the results of cytotoxicity by MTT assay, reduced viability of cells and cytotoxicity were observed. The 50% inhibitory concentration (IC50 of silver nanoparticles was recorded at 100 and 10 µg/ml for AgNP-A and AgNP-B, respectively. Moreover, microscopic observations indicated clear morphological changes of treated cells. Conclusion: Concentration and physicochemical properties of silver nanoparticles like size, shape, surface area, coating and zeta potential play a role in cytotoxicity and morphological changes of treated cells.

  5. Effect of 5-aminolevulinic acid on kinetics of protoporphyrin IX production in CHO cells.

    Directory of Open Access Journals (Sweden)

    W Warchoł

    2004-07-01

    Full Text Available 5-aminolevulinic acid (ALA is utilized in a photodynamic therapy as a compound capable of augmenting intracellular pool of protoporphyrin IX (PpIX, which exhibits properties of a photosensitizer. The studies were aimed at monitoring accumulation of endogenous protoporphyrin IX in CHO cells under effect of various concentrations of ALA in culture medium and following removal of the compound from the culture medium. Cell content of PpIX was determined following incubation of the cells for 72 h in a culture medium containing different concentration of ALA. Moreover, the cells were preincubated for 2 h in ALA at various concentrations and separated from the compound by medium change and their PpIX content was monitored following incubation. PpIX content was defined by a fluorescent technique under the confocal microscope. In the course of continuous incubation of cells with ALA, biphasic alterations were noted in cellular PpIX concentration. Removal of ALA from the incubation medium resulted at first in a decrease in PpIX content in cells, which was followed by an evidently augmented accumulation of the compound in the cells. The results suggested that in the case of CHO cells, exogenous ALA was not an exclusive source of PpIX synthesis and that alterations in enzyme activities were responsible for production of PpIX.

  6. Aerobic expression of Vitreoscilla hemoglobin improves the growth performance of CHO-K1 cells.

    Science.gov (United States)

    Juárez, Mariana; González-De la Rosa, Claudia H; Memún, Elisa; Sigala, Juan-Carlos; Lara, Alvaro R

    2017-03-01

    Inefficient carbon metabolism is a relevant issue during the culture of mammalian cells for the production of biopharmaceuticals. Therefore, cell engineering strategies to improve the metabolic and growth performance of cell lines are needed. The expression of Vitreoscilla stercoraria hemoglobin (VHb) has been shown to significantly reduce overflow metabolism and improve the aerobic growth of bacteria. However, the effects of VHb on mammalian cells have been rarely studied. Here, the impact of VHb on growth and lactate accumulation during CHO-K1 cell culture was investigated. For this purpose, CHO-K1 cells were transfected with plasmids carrying the vgb or gfp gene to express VHb or green fluorescence protein (GFP), respectively. VHb expression increased the specific growth rate and biomass yields on glucose and glutamine by 60 %, and reduced the amount of lactate produced per cell by 40 %, compared to the GFP-expression controls. Immunofluorescence microscopy showed that VHb is distributed in the cytoplasm and organelles, which support the hypothesis that VHb could serve as an oxygen carrier, enhancing aerobic respiration. These results are useful for the development of better producing cell lines for industrial applications. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin.

    Science.gov (United States)

    Baik, Jong Youn; Dahodwala, Hussain; Oduah, Eziafa; Talman, Lee; Gemmill, Trent R; Gasimli, Leyla; Datta, Payel; Yang, Bo; Li, Guoyun; Zhang, Fuming; Li, Lingyun; Linhardt, Robert J; Campbell, Andrew M; Gorfien, Stephen F; Sharfstein, Susan T

    2015-07-01

    Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity ∼100-fold and the heparin/heparan sulfate yield ∼10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in ∼two-fold increase in integrated viable cell density and a 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of ∼90 μg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. N-cadherin induces partial differentiation of cholinergic presynaptic terminals in heterologous cultures of brainstem neurons and CHO cells

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    Richard J Flannery

    2012-12-01

    Full Text Available N-cadherin is a calcium-sensitive cell adhesion molecule commonly expressed at synaptic junctions and contributes to formation and maturation of synaptic contacts. This study used heterologous cell cultures of brainstem cholinergic neurons and transfected Chinese Hamster Ovary (CHO cells to examine whether N-cadherin is sufficient to induce differentiation of cholinergic presynaptic terminals. Brainstem nuclei isolated from transgenic mice expressing EGFP under the control of choline acetyltransferase transcriptional regulatory elements (ChATBACEGFP were cultured as tissue explants for five days and cocultured with transfected CHO cells for an additional two days. Immunostaining for synaptic vesicle proteins SV2 and synapsin I revealed a ~3-fold increase in the area of SV2 immunolabeling over N-cadherin expressing CHO cells, and this effect was enhanced by coexpression of p120-catenin. Synapsin I immunolabeling per axon length was also increased on N-cadherin expressing CHO cells but required coexpression of p120-catenin. To determine whether N-cadherin induces formation of neurotransmitter release sites, whole-cell voltage-clamp recordings of CHO cells expressing alpha-3 and beta-4 nicotinic acetylcholine receptor (nAChR subunits in contact with cholinergic axons were used to monitor excitatory postsynaptic potentials (EPSPs and miniature EPSPs (mEPSPs. EPSPs and mEPSPs were not detected in both, control and in N-cadherin expressing CHO cells in the absence or presence of tetrodotoxin. These results indicate that expression of N-cadherin in non-neuronal cells is sufficient to initiate differentiation of presynaptic cholinergic terminals by inducing accumulation of synaptic vesicles; however, development of readily detectable mature cholinergic release sites and/or clustering of postsynaptic nAChR may require expression of additional synaptogenic proteins.

  9. A framework to quantify karyotype variation associated with CHO cell line instability at a single-cell level.

    Science.gov (United States)

    Baik, Jong Youn; Lee, Kelvin H

    2017-05-01

    Chinese hamster ovary (CHO) cells, the major mammalian host cells for biomanufacturing of therapeutic proteins, have been extensively investigated to enhance productivity and product quality. However, cell line instability resulting in unexpected changes in productivity or product quality continues to be a challenge. Based on previous reports about causes and characteristics of production instability, we hypothesized that chromosomal rearrangements due to genomic instability are associated with production instability and that these events can be characterized. We developed a production instability model using secreted alkaline phosphatase (SEAP)-expressing CHO cells (CHO-SEAP) as well as a framework to quantify chromosomal rearrangements by karyotyping. In the absence of methotrexate (MTX), CHO-SEAP cells exhibited a slightly increased growth rate, a significantly decreased specific productivity, and changes in the chromosomal rearrangement ratio of seven chromosomes. In contrast, when MTX was re-introduced, the growth rate and SEAP productivity reversed to the initial values, demonstrating the reversibility of production instability in CHO-SEAP cells. Fluorescence in situ hybridization analysis identified that the SEAP genes were incorporated in the chromosomal rearrangement (insertion) part of the der(Z9) chromosome. Karyotype analysis indicated that the insertion ratio of the der(Z9) chromosome decreased in the CHO-SEAP cells grown without MTX, demonstrating a correlation between chromosomal rearrangement and production instability. Our results support a mechanism for production instability, wherein a randomly generated chromosomal rearrangement (or genotype) results in cells with a growth advantage that is also associated with non (or low)-producing traits. As a result, the non-producing cells grow faster and thereby outgrow the producing population. Biotechnol. Bioeng. 2017;114: 1045-1053. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. Glycoprofiling effects of media additives on IgG produced by CHO cells in fed-batch bioreactors

    DEFF Research Database (Denmark)

    Kildegaard, Helene Faustrup; Fan, Yuzhou; Wagtberg Sen, Jette

    2016-01-01

    Therapeutic monoclonal antibodies (mAbs) are mainly produced by heterogonous expression in Chinese hamster ovary (CHO) cells. The glycosylation profile of the mAbs has major impact on the efficacy and safety of the drug and is therefore an important parameter to control during production. In this......Therapeutic monoclonal antibodies (mAbs) are mainly produced by heterogonous expression in Chinese hamster ovary (CHO) cells. The glycosylation profile of the mAbs has major impact on the efficacy and safety of the drug and is therefore an important parameter to control during production...

  11. Comparison of the Production of Recombinant Protein in Suspension Culture of CHO Cells in Spinner Flask and Shake Flask System

    Directory of Open Access Journals (Sweden)

    S.N.Z Zainul Abidin

    2011-12-01

    Full Text Available Chinese hamster ovary (CHO cells have been most widely used as the production host for the commercial production of biopharmaceuticals product. They have been extensively studied and developed, and today provide a stable platform for producing monoclonal antibodies and recombinant proteins. This study was focusing on comparison of suspension culture system by using spinner flask and shake flask for the growth and production of recombinant protein in CHO cell line. The CHO cells were transfected with an expression of DNA plasmid containing lac Z gene which codes for β-galactosidase. The recombinant genes in these CHO cells and the β-galactosidase expressing cells were adapted to suspension culture. The agitation speed for both spinner and shake flask were adjusted accordingly. The experiments were carried out in duplicate and samples were taken for cell count, determination of glucose consumption, lactate production and protein level by using biochemical assay. The result showed that, the cell growth in spinner flask is more favorable then in shake flask. The cell concentration in spinner flask is 58% higher than in shake flask. On the other hand, specific activity of β-galactosidase is 25% higher in spinner flask compared to shake flask, at the same agitation speed.ABSTRAK: Sel ovari hamster China (Chinese hamster ovary (CHO digunakan secara meluas dalam hos pembiakan untuk tujuan komersil produk biofarmaseutikal. Ia telah dikaji dan dibangunkan secara ekstensif, dan kini ia menyediakan landasan yang stabil untuk penghasilan antibodi monoklon dan protein rekombinan. Kajian ini memfokuskan tentang penghasilan protein rekombinan menggunakan kultur ampaian sel CHO di dalam kelalang putar dan kelalang goncang. Sel CHO dimasukkan dengan plasmid DNA yang mengandungi gen lac Z yang juga memberikan kod untuk β-galaktosidase. Sel CHO β-galaktosidase-terungkap dimasukkan ke dalam kultur ampaian. Kelajuan agitasi untuk kedua-dua kelalang putar

  12. Implicit Real Vector Automata

    Directory of Open Access Journals (Sweden)

    Jean-François Degbomont

    2010-10-01

    Full Text Available This paper addresses the symbolic representation of non-convex real polyhedra, i.e., sets of real vectors satisfying arbitrary Boolean combinations of linear constraints. We develop an original data structure for representing such sets, based on an implicit and concise encoding of a known structure, the Real Vector Automaton. The resulting formalism provides a canonical representation of polyhedra, is closed under Boolean operators, and admits an efficient decision procedure for testing the membership of a vector.

  13. Vectors and their applications

    CERN Document Server

    Pettofrezzo, Anthony J

    2005-01-01

    Geared toward undergraduate students, this text illustrates the use of vectors as a mathematical tool in plane synthetic geometry, plane and spherical trigonometry, and analytic geometry of two- and three-dimensional space. Its rigorous development includes a complete treatment of the algebra of vectors in the first two chapters.Among the text's outstanding features are numbered definitions and theorems in the development of vector algebra, which appear in italics for easy reference. Most of the theorems include proofs, and coordinate position vectors receive an in-depth treatment. Key concept

  14. Benchmarking of commercially available CHO cell culture media for antibody production.

    Science.gov (United States)

    Reinhart, David; Damjanovic, Lukas; Kaisermayer, Christian; Kunert, Renate

    2015-06-01

    In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but high amino acid concentrations alone were not sufficient to ensure superior cell growth and high antibody production. However, some key amino acids that were limiting in most media could be identified. Unbalanced glucose and amino acids led to high cell-specific lactate and ammonium production rates. In some media, persistently high glucose concentrations probably induced the suppression of respiration and oxidative phosphorylation, known as Crabtree effect, which resulted in high cell-specific glycolysis rates along with a continuous and high lactate production. In additional experiments, two of the eight basal media were supplemented with feeds from two different manufacturers in six combinations, in order to understand the combined impact of media and feeds on cell metabolism in a CHO fed-batch process. Cell growth, nutrient consumption and metabolite production rates, antibody production, and IgG quality were evaluated in detail. Concentrated feed supplements boosted cell concentrations almost threefold and antibody titers up to sevenfold. Depending on the fed-batch strategy, fourfold higher peak cell concentrations and eightfold increased IgG titers (up to 5.8 g/L) were achieved. The glycolytic flux was remarkably similar among the fed-batches; however, substantially different specific lactate production rates were observed in the different media and feed combinations. Further analysis revealed that in addition to the feed additives, the basal medium can make a considerable

  15. N-Glycosylation optimization of recombinant antibodies in CHO cell through process and metabolic engineering

    DEFF Research Database (Denmark)

    Fan, Yuzhou

    protein with ensured safety, efficacy and cost-effectiveness, holistic understanding of titer and N-glycosylation of the protein in relation to cell culture process as well as genomic, proteomic, metabolic and physiological status of the cells becomes a superior approach. Combining the knowledge of CHO...... cell culture technology, upstream process engineering, metabolic engineering, and glycobiology into a systematic framework allow us to improve the production of recombinant therapeutic protein towards an optimal balance between quantity and quality. In the presented work, recent know-how on impact...... monoclonal antibody (mAb) towards desired patterns, and at the same time try to understand the underlying mechanisms of that from a systems biology perspective. Two different strategies were used and achieved great success in glyco-optimization: 1) optimize media and culture process; 2) Genetically optimize...

  16. Genetic engineering of CHO cells for viral resistance to minute virus of mice.

    Science.gov (United States)

    Mascarenhas, Joaquina X; Korokhov, Nikolay; Burger, Lisa; Kassim, Ademola; Tuter, Jason; Miller, Daniel; Borgschulte, Trissa; George, Henry J; Chang, Audrey; Pintel, David J; Onions, David; Kayser, Kevin J

    2017-03-01

    Contamination by the parvovirus minute virus of mice (MVM) remains a challenge in Chinese hamster ovary (CHO) biopharmaceutical production processes. Although infrequent, infection of a bioreactor can be catastrophic for a manufacturer, can impact patient drug supply and safety, and can have regulatory implications. We evaluated engineering a CHO parental cell line (CHOZN® GS-/- ) to create a new host cell line that is resistant to MVM infection by modifying the major receptors used by the virus to enter cells. Attachment to a cell surface receptor is a key first step in the infection cycle for many viruses. While the exact functional receptor for MVM binding to CHO cell surface is unknown, sialic acid on the cell surface has been implicated. In this work, we used the zinc finger nuclease gene editing technology to validate the role of sialic acid on the cell surface in the binding and internalization of the MVM virus. Our approach was to systematically mutate genes involved in cell surface sialylation and then challenge each cell line for their ability to resist viral entry and propagation. To test the importance of sialylation, the following genes were knocked out: the CMP-sialic acid transporter, solute carrier family 35A1 (Slc35a1), the core 1-β-1,3-galactosyltransferase-1 specific chaperone (Cosmc), and mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (Mgat1) as well as members of the sialyltransferase family. Slc35a1 is responsible for transporting sialic acid into the Golgi. Knocking out function of this gene in a cell results in asialylated glycan structures, thus eliminating the ability of MVM to bind to and enter the cell. The complete absence of sialic acid on the Slc35a1 knockout cell line led to complete resistance to MVM infection. The Cosmc and Mgat1 knockouts also show significant inhibition of infection likely due to their effect on decreasing cell surface sialic acid. Previously in vitro glycan analysis has been used to

  17. Cell survival and chromosomal aberrations in CHO-K1 cells irradiated by carbon ions

    Energy Technology Data Exchange (ETDEWEB)

    Czub, J. [Institute of Physics, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Banas, D. [Institute of Physics, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Holycross Cancer Center, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Blaszczyk, A. [Faculty of Physics, Astronomy and Informatics, Nicolaus Copernicus University, Grudziadzka 5, 87-100 Torun (Poland); Braziewicz, J. [Institute of Physics, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Holycross Cancer Center, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Buraczewska, I. [Institute of Nuclear Chemistry and Technology, ul. Dorodna 16, 03-195 Warsaw (Poland); Choinski, J. [Heavy Ion Laboratory, Warsaw University, ul. Pasteura 5A, 02-093 Warsaw (Poland); Gorak, U. [Institute of Experimental Physics, Warsaw University, ul. Hoza 69, 00-681 Warsaw (Poland); Jaskola, M.; Korman, A. [Andrzej Soltan Institute for Nuclear Studies, 05-400 Otwock-Swierk (Poland); Lankoff, A.; Lisowska, H. [Institute of Biology, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Lukaszek, A. [Institute of Experimental Physics, Warsaw University, ul. Hoza 69, 00-681 Warsaw (Poland); Main School of Fire Service, ul. Slowackiego 52/54, 01-629 Warsaw (Poland); Szeflinski, Z. [Institute of Experimental Physics, Warsaw University, ul. Hoza 69, 00-681 Warsaw (Poland)], E-mail: szef@fuw.edu.pl; Wojcik, A. [Institute of Nuclear Chemistry and Technology, ul. Dorodna 16, 03-195 Warsaw (Poland); Institute of Biology, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland)

    2009-03-15

    Chinese hamster ovary CHO-K1 cells were exposed to high LET {sup 12}C-beam (LET: 830 keV/{mu}m) in the dose range of 0-6 Gy and to {sup 60}Co irradiation and the RBE value was obtained. Effects of {sup 12}C-beam exposure on cell survival and chromosomal aberrations were calculated. The chromosomal aberration data were fitted with linear equation. The distribution of aberration in cells was examined with a standard u-test and used to evaluate the data according to Poisson probabilities. The variance to the mean ratio {sigma}{sup 2}/Y and the dispersion index (u) were determined. Overdispersion was significant (p<0.05) when the value of u exceeded 1.96.

  18. Vector mesons in matter

    Indian Academy of Sciences (India)

    One consequence of the chiral restoration is the mixing of parity partners. We look for a possible signature of the mixing of vector and axial vector mesons in heavy-ion collisions. We suggest an experimental method for its observation. The dynamical evolution of the heavy-ion collision is described by a transport equation of ...

  19. Vector mesons in matter

    Indian Academy of Sciences (India)

    kfki.hu. Abstract. One consequence of the chiral restoration is the mixing of parity partners. We look for a possible signature of the mixing of vector and axial vector mesons in heavy- ion collisions. We suggest an experimental method for its ...

  20. Solution and solid-state models of peptide CH...O hydrogen bonds.

    Science.gov (United States)

    Baures, Paul W; Beatty, Alicia M; Dhanasekaran, Muthu; Helfrich, Brian A; Pérez-Segarra, Waleska; Desper, John

    2002-09-25

    Fumaramide derivatives were analyzed in solution by (1)H NMR spectroscopy and in the solid state by X-ray crystallography in order to characterize the formation of CH...O interactions under each condition and to thereby serve as models for these interactions in peptide and protein structure. Solutions of fumaramides at 10 mM in CDCl(3) were titrated with DMSO-d(6), resulting in chemical shifts that moved downfield for the CH groups thought to participate in CH...O=S(CD(3))(2) hydrogen bonds concurrent with NH...O=S(CD(3))(2) hydrogen bonding. In this model, nonparticipating CH groups under the same conditions showed no significant change in chemical shifts between 0.0 and 1.0 M DMSO-d(6) and then moved upfield at higher DMSO-d(6) concentrations. At concentrations above 1.0 M DMSO-d(6), the directed CH...O=S(CD(3))(2) hydrogen bonds provide protection from random DMSO-d(6) contact and prevent the chemical shifts for participating CH groups from moving upfield beyond the original value observed in CDCl(3). X-ray crystal structures identified CH...O=C hydrogen bonds alongside intermolecular NH...O=C hydrogen bonding, a result that supports the solution (1)H NMR spectroscopy results. The solution and solid-state data therefore both provide evidence for the presence of CH...O hydrogen bonds formed concurrent with NH...O hydrogen bonding in these structures. The CH...O=C hydrogen bonds in the X-ray crystal structures are similar to those described for antiparallel beta-sheet structure observed in protein X-ray crystal structures.

  1. Fluorescence dye-based detection of mAb aggregates in CHO culture supernatants.

    Science.gov (United States)

    Paul, Albert Jesuran; Schwab, Karen; Prokoph, Nina; Haas, Elena; Handrick, René; Hesse, Friedemann

    2015-06-01

    Product yields, efficacy, and safety of monoclonal antibodies (mAbs) are reduced by the formation of higher molecular weight aggregates during upstream processing. In-process characterization of mAb aggregate formation is a challenge since there is a lack of a fast detection method to identify mAb aggregates in cell culture. In this work, we present a rapid method to characterize mAb aggregate-containing Chinese hamster ovary (CHO) cell culture supernatants. The fluorescence dyes thioflavin T (ThT) and 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS) enabled the detection of soluble as well as large mAb aggregates. Partial least square (PLS) regression models were used to evaluate the linearity of the dye-based mAb aggregate detection in buffer down to a mAb aggregate concentration of 2.4 μg mL(-1). Furthermore, mAb aggregates were detected in bioprocess medium using Bis-ANS and ThT. Dye binding to aggregates was stable for 60 min, making the method robust and reliable. Finally, the developed method using 10 μmol L(-1) Bis-ANS enabled discrimination between CHO cell culture supernatants containing different levels of mAb aggregates. The method can be adapted for high-throughput screening, e.g., to screen for cell culture conditions influencing mAb product quality, and hence can contribute to the improvement of production processes of biopharmaceuticals in mammalian cell culture.

  2. Quantitative mammalian cell mutagenesis and mutagen screening: study with CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Hsie, A.W.; O' Neill, J.P.; San Sebastian, J.R.; Brimer, P.A.

    1979-01-01

    The CHO/HGPRT system has been developed and defined for quantifying mutation induced by various physical and chemical agents at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells. In all direct-acting chemical mutagens studied, mutation induction increases linearly as a function of the concentration, with no apparent threshold. Some chemicals induce mutation at non-cytotoxic concentrations. The mutagenicity of ethyl methanesulfonate has been quantified as a function of exposure concentration x treatment time. The sensitive and quantitative nature of the system enables studies of the structure-activity (mutagenicity) relationships of various classes of chemicals, including alkylating agents, heterocyclic nitrogen mustards, and platinum compounds. When rat liver S/sub 9/-mediated metabolic activation is present, procarcinogens such as benzo(a)pyrene, 2-acetylaminofluorene, and dimethylnitrosamine are mutagenic, whereas their noncarcinogenic structural analogues pyrene, fluorene, and dimethylamine are not. The system has been shown to be useful in determining the interactive effects between physical and chemical agents, and in screening for mutagenicity of fractionated organic mixtures and industrial chemicals in both liquid and gaseous state. For the system to be used successfully in routine screening, further studies should be directed toward the development of a metabolic activation system suitable for a broad spectrum of chemicals, a sensitive and reliable statistical method, and an experimental design to determine compounds with low mutagenicity. The system has been expanded for determination of mutagen-induced chromosome aberration, sister-chromatid exchange, and micronucleus formation in addition to gene mutation and cytotoxicity; it can also be used to study inhibition of DNA synthesis. (ERB)

  3. Complex Polynomial Vector Fields

    DEFF Research Database (Denmark)

    Dias, Kealey

    The two branches of dynamical systems, continuous and discrete, correspond to the study of differential equations (vector fields) and iteration of mappings respectively. In holomorphic dynamics, the systems studied are restricted to those described by holomorphic (complex analytic) functions...... or meromorphic (allowing poles as singularities) functions. There already exists a well-developed theory for iterative holomorphic dynamical systems, and successful relations found between iteration theory and flows of vector fields have been one of the main motivations for the recent interest in holomorphic...... vector fields. Since the class of complex polynomial vector fields in the plane is natural to consider, it is remarkable that its study has only begun very recently. There are numerous fundamental questions that are still open, both in the general classification of these vector fields, the decomposition...

  4. Impact of CHO Metabolism on Cell Growth and Protein Production: An Overview of Toxic and Inhibiting Metabolites and Nutrients

    DEFF Research Database (Denmark)

    Pereira, Sara; Kildegaard, Helene F.; Andersen, Mikael R.

    2018-01-01

    For over three decades, Chinese hamster ovary (CHO) cells have been the chosen expression platform for the production of therapeutic proteins with complex post-translational modifications. However, the metabolism of these cells is far from perfect and optimized, and requires substantial knowhow...

  5. Engineer medium and feed for modulating N-glycosylation of recombinant protein production in CHO cell culture

    DEFF Research Database (Denmark)

    Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2017-01-01

    Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N...

  6. Amino Acid Mixture Acts as a Potent VEGF Lowering Agent in CHO-K1 Cells Exposed to High Glucose.

    Science.gov (United States)

    Selvi, Radhakrishnan; Bhuvanasundar, Renganathan; Angayarkanni, Narayanasamy

    2017-04-01

    Though the role of amino acids in Diabetes Mellitus is controversial, the beneficial effect of amino acids in Diabetes Mellitus has been reported based on its anti-glycating property and insulin potentiating effects. In the current study, we evaluated the ROS generation and VEGF expression in CHO-K1 cells induced by high glucose concentration. The effect of amino acids treatment was studied under this condition to evaluate the VEGF lowering effect. CHO-K1 cells were treated various concentration of glucose (7 mmol, 17 mmol and 27 mmol) with and without free amino acids (5 mmol) or the amino acids mixture (AAM). Intracellular reactive oxygen species (ROS) was estimated by fluorescein dye (DCFDA), nitric oxide (NO) by Griess reaction, hydrogen peroxide (H2O2) by fluorimetry using Amplex red dye, super oxide dismutase (SOD) by spectrophotometry and VEGF by immunoblotting. High glucose condition significantly induced the expression of VEGF and this was reduced significantly by AAM treatment (p = 0.004). AAM also significantly decreased the cellular levels of ROS, NO, H2O2 as well as the SOD activity in CHO-K1 cells exposed to high glucose condition (p <0.05). The present study identified AAM as a potential VEGF lowering agent that intervenes at the level of oxidative stress in high glucose conditions as evaluated in CHO-K1 cells. Copyright © 2017 IMSS. Published by Elsevier Inc. All rights reserved.

  7. miR-92a enhances recombinant protein productivity in CHO cells by increasing intracellular cholesterol levels.

    Science.gov (United States)

    Loh, Wan Ping; Yang, Yuansheng; Lam, Kong Peng

    2017-04-01

    MicroRNAs (miRNAs) have emerged as promising targets for engineering of CHO cell factories to enhance recombinant protein productivity. Manipulation of miRNA levels in CHO cells have been shown to improve product yield by increasing proliferation and specific productivity (qP), resisting apoptosis and enhancing oxidative metabolism. The authors previously demonstrated that over-expressing miR-92a results in increases in qP and titer of CHO-IgG cells. However, the mechanisms by which miR-92a enhances qP in CHO cells are still uninvestigated. Here, the authors report the identification of insig1, a regulator of cholesterol biosynthesis, as a target of miR-92a using computational prediction. Both transient and stable over-expression of miR-92a decreased the expression levels of insig1. Insig1 was further validated as a target of miR-92a using 3' UTR reporter assay. Intracellular cholesterol concentration of two high-producing miR-92a clones were significantly increased by ≈30% compared to the blank-transfected pool. Relative Golgi surface area was also found to be 18-26% higher in these clones. Our findings suggest that miR-92a may affect cholesterol metabolism by repressing insig1, resulting in raised intracellular cholesterol levels and Golgi volume and hence enhanced protein secretion. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Impact of sodium butyrate and mild hypothermia on metabolic and physiological behaviour of CHO TF 70R cells

    Directory of Open Access Journals (Sweden)

    Veronica Avello

    2017-05-01

    Conclusions: The combination of NaBu addition and mild hypothermic condition causes an impact on physiological and metabolic state of CHO TF 70R cells, decreasing cell growth rate and improving glucose consumption efficiency. These results therefore provide a promising strategy to increase specific productivity of rh-tPA.

  9. Accelerating Genome Editing in CHO Cells Using CRISPR Cas9 and CRISPy, a Web-Based Target Finding Tool

    DEFF Research Database (Denmark)

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Hansen, Henning Gram

    2014-01-01

    by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genomeediting methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift...

  10. Vector SIMP dark matter

    Science.gov (United States)

    Choi, Soo-Min; Hochberg, Yonit; Kuflik, Eric; Lee, Hyun Min; Mambrini, Yann; Murayama, Hitoshi; Pierre, Mathias

    2017-10-01

    Strongly Interacting Massive Particles (SIMPs) have recently been proposed as light thermal dark matter relics. Here we consider an explicit realization of the SIMP mechanism in the form of vector SIMPs arising from an SU(2) X hidden gauge theory, where the accidental custodial symmetry protects the stability of the dark matter. We propose several ways of equilibrating the dark and visible sectors in this setup. In particular, we show that a light dark Higgs portal can maintain thermal equilibrium between the two sectors, as can a massive dark vector portal with its generalized Chern-Simons couplings to the vector SIMPs, all while remaining consistent with experimental constraints.

  11. Vector Difference Calculus

    Science.gov (United States)

    Schwalm, W. A.; Schwalm, M. K.; Giona, M.

    1998-03-01

    Space is filled with triangulating graph \\calG to serve as a quadrature grid. A discrete analog of the theory of differential forms is constructed using the associated simplical complex. The role of a basis for Λ^p at a point is played by the set of (p+1) -simplices containing a given vertex. Vector difference operations analogous to div, grad and curl, together with corresponding vector identities and exact difference analogs of the Stokes-type theorems, are obtained in terms of the boundary partial and coboundary d. Difference versions of the full vector Maxwell electromagnetic equations are analyzed on a random structure.

  12. GAP Land Cover - Vector

    Data.gov (United States)

    Minnesota Department of Natural Resources — This vector dataset is a detailed (1-acre minimum), hierarchically organized vegetation cover map produced by computer classification of combined two-season pairs of...

  13. Tagged Vector Contour (TVC)

    Data.gov (United States)

    Kansas Data Access and Support Center — The Kansas Tagged Vector Contour (TVC) dataset consists of digitized contours from the 7.5 minute topographic quadrangle maps. Coverage for the state is incomplete....

  14. Syngeneic AAV pseudo-vectors potentiates full vector transduction

    Science.gov (United States)

    An excessive amount of empty capsids are generated during regular AAV vector production process. These pseudo-vectors often remain in final vectors used for animal studies or clinical trials. The potential effects of these pseudo-vectors on AAV transduction have been a major concern. In the current ...

  15. Vector and axial vector mesons at finite temperature

    OpenAIRE

    mallik, S.; Sarkar, Sourav

    2002-01-01

    We consider the thermal correlation functions of vector and axial-vector currents and evaluate corrections to the vector and axial-vector meson pole terms to one loop in chiral perturbation theory. As expected, the pole positions do not shift to leading order in temperature. But the residues decrease with temperature.

  16. Leveraging a CHO cell line toolkit to accelerate biotherapeutics into the clinic.

    Science.gov (United States)

    Wright, Chapman; Alves, Christina; Kshirsagar, Rashmi; Pieracci, John; Estes, Scott

    2017-11-01

    The Biogen upstream platform is capable of delivering equivalent quality material throughout the cell line generation process. This allows us to rapidly deliver high-quality biopharmaceuticals to patients with unmet medical needs. The drive to reduce time-to-market led the cell engineering group to develop an expression system that can enable this strategy. We have developed a clonal Chinese Hamster Ovary (CHO) host cell line that can routinely produce consistent antibody material at high titers throughout the cell line generation process. This host line enables faster delivery of early phase material through use of the highly productive stable pool or a mixture of high performance clones. Due to unique characteristics of this cell line, the product quality of material from early cell populations is very comparable to material from the final clones. This lends itself to a "fast-to-tox" strategy whereby toxicology studies can be performed with representative material from an earlier cell population, thus accelerating the clinical timelines. Our new clonal host offers robust and consistent performance that enables a highly productive, flexible process and faster preclinical timelines. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1468-1475, 2017. © 2017 American Institute of Chemical Engineers.

  17. Characterization of recombinant human diamine oxidase (rhDAO) produced in Chinese Hamster Ovary (CHO) cells.

    Science.gov (United States)

    Gludovacz, Elisabeth; Maresch, Daniel; Bonta, Maximilian; Szöllösi, Helen; Furtmüller, Paul G; Weik, Robert; Altmann, Friedrich; Limbeck, Andreas; Borth, Nicole; Jilma, Bernd; Boehm, Thomas

    2016-06-10

    Human diamine oxidase (hDAO) efficiently degrades polyamines and histamine. Reduced enzyme activities might cause complications during pregnancy and be involved in histamine intolerance. So far hDAO has been characterized after isolation from either native sources or the heterologous production in insect cells. Accessibility to human enzyme is limited and insect cells produce non-human glycosylation patterns that may alter its biochemical properties. We present the heterologous expression of hDAO in Chinese Hamster Ovary (CHO) cells and a three step purification protocol. Analysis of metal content using ICP-MS revealed that 93% of the active sites were occupied by copper. Topaquinone (TPQ) cofactor content was determined using phenylhydrazine titration. Ninety-four percent of DAO molecules contained TPQ and therefore the copper content at the active site was indirectly confirmed. Mass spectrometric analysis was conducted to verify sequence integrity of the protein and to assess the glycosylation profile. Electronic circular dichroism and UV-vis spectra data were used to characterize structural properties. The substrate preference and kinetic parameters were in accordance with previous publications. The establishment of a recombinant production system for hDAO enables us to generate decent amounts of protein with negligible impurities to address new scientific questions. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Measurement and control of host cell proteins (HCPs) in CHO cell bioprocesses.

    Science.gov (United States)

    Hogwood, Catherine E M; Bracewell, Daniel G; Smales, C Mark

    2014-12-01

    Chinese hamster ovary (CHO) cells are widely used for the production of biotherapeutic recombinant proteins for a range of molecules including monoclonal antibodies and Fc-fusion proteins. Regulatory requirements for the final product include the removal of host cell proteins (HCPs) to acceptable amounts (<100ppm). Recent research has begun to unravel the extent to which upstream process conditions and subsequent product recovery and purification processes impact upon the HCP profile. A number of upstream parameters, including the selection of the cell line, the culturing process (e.g. feeding regime, culture temperature), cell viability at time of harvest/culture duration and cell shear sensitivity can all influence the resulting HCP profile. Further, the molecule itself plays an important role in determining those HCPs that are retained throughout a bioprocess and HCPs can co-elute with the target product during purification. Measurement and monitoring of HCPs is usually undertaken using ELISA technology, however alternative approaches are also now emerging that complement ELISA and allow the detection, identification and monitoring of specific HCPs. Here we discuss our understanding of how the process itself influences those HCPs present throughout the production process and the challenges in their monitoring, measurement and removal. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Enhanced production of monomeric interferon-beta by CHO cells through the control of culture conditions.

    Science.gov (United States)

    Rodriguez, J; Spearman, M; Huzel, N; Butler, M

    2005-01-01

    The enhancement of recombinant protein expression of a transfected cell line is essential for the development of an efficient large-scale bioprocess. The effect of various media additives and temperature conditions were studied in an attempt to optimize protein production, stability, and protein glycosylation from a Chinese hamster ovary (CHO) cell line producing human beta-interferon (Hu-beta-IFN). We observed a decrease in the ELISA response of the glycoprotein in the later stages of batch cultures, which was attributed to molecular aggregation. Cells were subjected to various concentrations of glycerol, dimethyl sulfoxide (DMSO), and sodium butyrate (NaBu) in a variety of culture systems and conditions. The addition of both NaBu and DMSO resulted in higher specific productivities but reduced growth rates that resulted in a net reduction of interferon produced. Glycerol appeared to stabilize the secreted beta-IFN, resulting in reduced aggregation, despite a decrease in cell growth rate. Glycosylation analysis of isolated beta-IFN showed a time-dependent decrease in sialylation in batch culture that was ameliorated by the presence of glycerol. Low-temperature conditions (30 degrees C) had the greatest effect on productivity with a significant increase in beta-IFN titer as well as a reduction in the degree of molecular aggregation.

  20. Cytotoxic effects induced by patulin, sterigmatocystin and beauvericin on CHO-K1 cells.

    Science.gov (United States)

    Zouaoui, Nidhal; Mallebrera, Beatriz; Berrada, Houda; Abid-Essefi, Salwa; Bacha, Hassen; Ruiz, Maria-Jose

    2016-03-01

    Mycotoxins are produced by different genera of fungi; mainly Aspergillus, Penicillium and Fusarium. The natural co-occurrence of beauvericin (BEA), patulin (PAT) and sterigmatocystin (STE) has been proved in feed and food commodities. This study investigates the cytotoxicity of individual and combined mycotoxins BEA, PAT and STE. The cytotoxicity on immortalized ovarian cells (CHO-K1) was evaluated using the MTT assay. After 24, 48 and 72 h, the IC50 values were 2.9 μM for PAT and ranged from 10.7 to 2.2 μM and from 25.0 to 12.5 μM for BEA and STE, respectively. Cytotoxic interactions were assayed by the isobologram method, which provides a combination index (CI) value as a quantitative measure of the three mycotoxin interaction's degree. Binary and tertiary combinations showed a dose dependent effect. At low fraction affected, mycotoxin combinations were synergetic; whereas, at higher fraction affected, the combinations showed additive effect. Our results indicate that the co-occurrence of low concentrations of mycotoxin in food may increase their toxic effects. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. An in-silico study of the regulation of CHO cells glycolysis.

    Science.gov (United States)

    Ghorbaniaghdam, Atefeh; Henry, Olivier; Jolicoeur, Mario

    2014-09-21

    In this work, a kinetic-metabolic model previously developed for CHO cells is used to study glycolysis regulation. The model is assessed for its biological relevance by analyzing its ability to simulate metabolic events induced following a hypoxic perturbation. Feedback and feedforward regulatory mechanisms known to occur to either inhibit or activate fluxes of glycolysis, are implemented in various combined scenarios and their effects on the metabolic response were analyzed. This study aims at characterizing the role of intermediates of glycolysis and of the cell energetic state, described as the AMP-to-ATP ratio, as inhibitors and activators of glycolysis pathway. In addition to the glycolysis pathway, we here describe the transient metabolic response of pathways that are connected to glycolysis, such as the pentose phosphate pathway, TCA cycle, cell bioenergetics system, glutamine and amino acids metabolisms. Taken individually, each regulatory mechanism leads to an oscillatory behavior in response to a hypoxic perturbation, while their combination clearly damps oscillations. However, only the addition of the cell energetic state to the regulatory mechanisms results in a non-oscillating response leading to metabolic flux rate rearrangement corresponding to the anaerobic metabolism expected to prevail under hypoxic conditions. We thus demonstrate in this work, from model simulations, that the robustness of a cell energetic metabolism can be described from a combination of feedback and feedforward inhibition and activation regulatory mechanisms of glycolysis fluxes, involving intermediates of glycolysis and the cell energetic state itself. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Genotoxicity and cytotoxicity of glass ionomer cements on Chinese hamster ovary (CHO) cells.

    Science.gov (United States)

    Ribeiro, Daniel Araki; Marques, Mariangela Esther Alencar; Salvadori, Daisy Maria Favero

    2006-06-01

    Glass ionomer cements are widely used in dentistry as restorative materials and adhesives for composite restorations. However, the results of genotoxicity studies using these materials are inconclusive in literature. The goal of this study was to examine the genotoxic and cytotoxic potential of three different glass ionomer cements available commercially (Ketac Cem, Ketac Molar and Vitrebond) by the single cell gel (comet) assay and trypan blue exclusion test, respectively. For this, such materials were exposed to Chinese hamster ovary (CHO) cells in vitro for 1 h at 37( composite function)C. Data were assessed by Kruskall-Wallis nonparametric test. The results showed that the powder from Ketac Molar displayed genotoxicity only in the maximum concentration evaluated (100 microg/mL). In the same way, the liquid from Vitrebond at 0.1% dilution caused an increase of DNA injury. Significant differences (P<0.05) in cytotoxicity provoked by all powders tested of glass ionomer cements were observed for exposure at 1,000 microg/mL concentration. With respect to liquids of glass ionomer cements evaluated, the major toxic effect on cell viability was produced at 10%, beginning at the dilution of 0.5% for Vitrebond. Taken together, we conclude that some components of glass ionomer cements show both genotoxic and cytotoxic effects.

  3. Mutagenicity of silver nanoparticles in CHO cells dependent on particle surface functionalization and metabolic activation

    Science.gov (United States)

    Guigas, Claudia; Walz, Elke; Gräf, Volker; Heller, Knut J.; Greiner, Ralf

    2017-06-01

    The potential of engineered nanomaterials to induce genotoxic effects is an important aspect of hazard identification. In this study, cytotoxicity and mutagenicity as a function of metabolic activation of three silver nanoparticle (AgNP) preparations differing in surface coating were determined in Chinese hamster ovary (CHO) subclone K1 cells. Three silver nanoparticle preparations ( x 90,0 polyvinylpyrrolidone (PVP-Ag) were used for the experiments. The cytotoxic effect of AgNPs was assessed with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide) test using different concentrations of nanoparticles, while the mutagenicity was evaluated using the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation assay. The cytotoxicity of all three AgNPs was lower in a cell culture medium containing 10% fetal calf serum (FCS) than in medium without FCS. The HPRT test without metabolic activation system S9 revealed that compared to the other AgNP formulations, citrate-coated Ag showed a lower genotoxic effect. However, addition of S9 increased the mutation frequency of all AgNPs and especially influenced the genotoxicity of Citrate-Ag. The results showed that exogenous metabolic activation of nanosilver is crucial even if interactions of the metabolic activation system, nanosilver, and cells are not really understood up to now.

  4. Near-threshold H/D exchange in CD₃CHO photodissociation.

    Science.gov (United States)

    Heazlewood, Brianna R; Maccarone, Alan T; Andrews, Duncan U; Osborn, David L; Harding, Lawrence B; Klippenstein, Stephen J; Jordan, Meredith J T; Kable, Scott H

    2011-06-01

    Measuring the isotopic abundance of hydrogen versus deuterium atoms is a key method for interrogating reaction pathways in chemistry. H/D 'scrambling' is the intramolecular rearrangement of labile isotopes of hydrogen atoms and when it occurs through unanticipated pathways can complicate the interpretation of such experiments. Here, we investigate H/D scrambling in acetaldehyde at the energetic threshold for breaking the formyl C-H bond and reveal an unexpected unimolecular mechanism. Laser photolysis experiments of CD₃CHO show that up to 17% of the products have undergone H/D exchange to give CD₂H + DCO. Transition-state theory calculations reveal that the dominant mechanism involves four sequential H- or D-shifts to form CD₂HCDO, which then undergoes conventional C-C bond cleavage. At the lowest energy the molecule undergoes an average of 20 H- or D-shifts before products are formed, evincing significant scrambling of H and D atoms. Analogous photochemically induced isomerizations and isotope scrambling are probably important in both atmospheric chemistry and combustion reactions.

  5. Extended Mixed Vector Equilibrium Problems

    Directory of Open Access Journals (Sweden)

    Mijanur Rahaman

    2014-01-01

    Full Text Available We study extended mixed vector equilibrium problems, namely, extended weak mixed vector equilibrium problem and extended strong mixed vector equilibrium problem in Hausdorff topological vector spaces. Using generalized KKM-Fan theorem (Ben-El-Mechaiekh et al.; 2005, some existence results for both problems are proved in noncompact domain.

  6. Engineer Medium and Feed for Modulating N-Glycosylation of Recombinant Protein Production in CHO Cell Culture.

    Science.gov (United States)

    Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2017-01-01

    Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N-glycosylation is crucial, as the structure of N-glycans can largely influence both biological and physicochemical properties of recombinant proteins. Protein N-glycosylation in CHO cell culture can be controlled and tuned by engineering medium, feed, culture process, as well as genetic elements of the cell. In this chapter, we will focus on how to carry out experiments for N-glycosylation modulation through medium and feed optimization. The workflow and typical methods involved in the experiment process will be presented.

  7. Channelized relevance vector machine as a numerical observer for cardiac perfusion defect detection task

    Science.gov (United States)

    Kalayeh, Mahdi M.; Marin, Thibault; Pretorius, P. Hendrik; Wernick, Miles N.; Yang, Yongyi; Brankov, Jovan G.

    2011-03-01

    In this paper, we present a numerical observer for image quality assessment, aiming to predict human observer accuracy in a cardiac perfusion defect detection task for single-photon emission computed tomography (SPECT). In medical imaging, image quality should be assessed by evaluating the human observer accuracy for a specific diagnostic task. This approach is known as task-based assessment. Such evaluations are important for optimizing and testing imaging devices and algorithms. Unfortunately, human observer studies with expert readers are costly and time-demanding. To address this problem, numerical observers have been developed as a surrogate for human readers to predict human diagnostic performance. The channelized Hotelling observer (CHO) with internal noise model has been found to predict human performance well in some situations, but does not always generalize well to unseen data. We have argued in the past that finding a model to predict human observers could be viewed as a machine learning problem. Following this approach, in this paper we propose a channelized relevance vector machine (CRVM) to predict human diagnostic scores in a detection task. We have previously used channelized support vector machines (CSVM) to predict human scores and have shown that this approach offers better and more robust predictions than the classical CHO method. The comparison of the proposed CRVM with our previously introduced CSVM method suggests that CRVM can achieve similar generalization accuracy, while dramatically reducing model complexity and computation time.

  8. CHO cell production and sequence improvement in the 13C6FR1 anti-Ebola antibody.

    Science.gov (United States)

    Pettit, Dean K; Rogers, Richard S; Arthur, Kelly; Brodsky, Yan; Clark, Rutilio H; Crowell, Chris; Ennis, Jane; Gillespie, Alison; Gillespie, Ron; Livingston, Brittney; Nalbandian, Edith; Pace, Danielle; Smidt, Pauline; Pauly, Michael; Timmons, Ken; Trentalange, Michael; Whaley, Kevin J; Zeitlin, Larry; Thomas, James N

    2016-01-01

    From March 2014 through February 2015, the Ebola virus spread rapidly in West Africa, resulting in almost 30,000 infections and approximately 10,000 deaths. With no approved therapeutic options available, an experimental antibody cocktail known as ZMapp™ was administered to patients on a limited compassionate-use basis. The supply of ZMapp™ was highly constrained at the time because it was in preclinical development and a novel production system (tobacco plants) was being used for manufacturing. To increase the production of ZMapp™ for an uncertain future demand, a consortium was formed in the fall of 2014 to quickly manufacture these anti-Ebola antibodies in Chinese hamster ovary (CHO) cells using bioreactors for production at a scale appropriate for thousands of doses. As a result of the efforts of this consortium, valuable lessons were learned about the processing of the antibodies in a CHO-based system. One of the ZMapp™ cocktail antibodies, known as c13C6FR1, had been sequence-optimized in the framework region for production in tobacco and engineered as a chimeric antibody. When transfected into CHO cells with the unaltered sequence, 13C6FR1 was difficult to process. This report describes efforts to produce 13C6FR1 and the parental murine hybridoma sequence, 13C6mu, in CHO cells, and provides evidence for the insertion of a highly conserved framework amino acid that improved the physical properties necessary for high-level expression and purification. Furthermore, it describes the technical and logistical lessons learned that may be beneficial in the event of a future Ebola virus or other pandemic viral outbreaks where mAbs are considered potential therapeutics.

  9. Cytotoxic effects of zearalenone and its metabolites and antioxidant cell defense in CHO-K1 cells.

    Science.gov (United States)

    Tatay, Elena; Font, Guillermina; Ruiz, Maria-Jose

    2016-10-01

    Zearalenone (ZEA) and its metabolites (α-zearalenol; α-ZOL, β-zearalenol; β-ZOL) are secondary metabolites of Fusarium fungi that produce cell injury. The present study explores mycotoxin-induced cell damage and cellular protection mechanisms in CHO-K1 cells. Cytotoxicity has been determined by reactive oxygen species (ROS) production and DNA damage. ROS production was determined using the fluorescein assay and DNA strand breakage by comet assay. Intracellular protection systems were glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD). The results demonstrated that all mycotoxins increased the ROS levels up to 5.3-fold the control levels in CHO-K1 cells. Zearalenone metabolites, but not ZEA, increased DNA damage 43% (α-ZOL) and 28% (β-ZOL) compared to control cells. The GSH levels decreased from 18% to 36%. The GPx and SOD activities respectively increased from 26% to 62% and from 23% to 69% in CHO-K1 cells, whereas CAT activity decreased from 14% to 52%. In addition, intracellular ROS production was induced by ZEA and its metabolites. The endogenous antioxidant system components GSH, GPx and SOD were activated against ZEA and its metabolites. These antioxidant system components thus could contribute to decrease cell injury by ZEA and its metabolites. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Internal noise in channelized Hotelling observer (CHO) study of detectability index-differential phase contrast CT vs. conventional CT

    Science.gov (United States)

    Tang, Xiangyang; Yang, Yi

    2014-03-01

    The channelized Hotelling observer (CHO) model, wherein internal noise plays an important role to account for the psychophysiological uncertainty in human's visual perception, has found extensive applications in the assessment of image quality in nuclear medicine, mammography and conventional CT. Recently, we extended its application to investigating the detectability index of differential phase contrast (DPC) CT-an emerging CT technology with the potential of increasing the capability in soft tissue differentiation. We found that the quantitative determination of internal noise in the CHO study of DPC-CT's detectability index should differ from that in the conventional CT. It is believed that the root cause of such a difference lies in the distinct noise spectra between the DPC-CT and conventional CT. In this paper, we present the preliminary results and investigate the adequate strategies to quantitatively determine the internal noise of CHO model for its application in the assessment of image quality in DPC-CT and its comparison with that of the conventional CT.

  11. Randomized Controlled Trial of Hospital-Based Hygiene and Water Treatment Intervention (CHoBI7) to Reduce Cholera.

    Science.gov (United States)

    George, Christine Marie; Monira, Shirajum; Sack, David A; Rashid, Mahamud-ur; Saif-Ur-Rahman, K M; Mahmud, Toslim; Rahman, Zillur; Mustafiz, Munshi; Bhuyian, Sazzadul Islam; Winch, Peter J; Leontsini, Elli; Perin, Jamie; Begum, Farzana; Zohura, Fatema; Biswas, Shwapon; Parvin, Tahmina; Zhang, Xiaotong; Jung, Danielle; Sack, R Bradley; Alam, Munirul

    2016-02-01

    The risk for cholera infection is >100 times higher for household contacts of cholera patients during the week after the index patient seeks hospital care than it is for the general population. To initiate a standard of care for this high-risk population, we developed Cholera-Hospital-Based-Intervention-for-7-Days (CHoBI7), which promotes hand washing with soap and treatment of water. To test CHoBI7, we conducted a randomized controlled trial among 219 intervention household contacts of 82 cholera patients and 220 control contacts of 83 cholera patients in Dhaka, Bangladesh, during 2013-2014. Intervention contacts had significantly fewer symptomatic Vibrio cholerae infections than did control contacts and 47% fewer overall V. cholerae infections. Intervention households had no stored drinking water with V. cholerae and 14 times higher odds of hand washing with soap at key events during structured observation on surveillance days 5, 6, or 7. CHoBI7 presents a promising approach for controlling cholera among highly susceptible household contacts of cholera patients.

  12. Enhanced genome editing tools for multi-gene deletion knock-out approaches using paired CRISPR sgRNAs in CHO cells

    DEFF Research Database (Denmark)

    Schmieder, Valerie; Bydlinski, Nina; Strasser, Richard

    2017-01-01

    Since the establishment of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9, powerful strategies for engineering of CHO cell lines have emerged. Nevertheless, there is still room to expand the scope of the CRISPR tool box for further applications to improve CHO cell factories....... Here, we demonstrate activity of the alternative CRISPR endonuclease Cpf1 in CHO-K1 for the first time and that it can be used in parallel to CRISPR/Cas9 without any interference. Both, Cas9 and Cpf1, can be effectively used for multi-gene engineering with a strategy based on paired single guide RNAs...... of application of CRISPR for novel gene editing approaches in CHO cells and also enable the efficient realization of a genome-wide deletion library....

  13. Bunyavirus-Vector Interactions

    Directory of Open Access Journals (Sweden)

    Kate McElroy Horne

    2014-11-01

    Full Text Available The Bunyaviridae family is comprised of more than 350 viruses, of which many within the Hantavirus, Orthobunyavirus, Nairovirus, Tospovirus, and Phlebovirus genera are significant human or agricultural pathogens. The viruses within the Orthobunyavirus, Nairovirus, and Phlebovirus genera are transmitted by hematophagous arthropods, such as mosquitoes, midges, flies, and ticks, and their associated arthropods not only serve as vectors but also as virus reservoirs in many cases. This review presents an overview of several important emerging or re-emerging bunyaviruses and describes what is known about bunyavirus-vector interactions based on epidemiological, ultrastructural, and genetic studies of members of this virus family.

  14. Free topological vector spaces

    OpenAIRE

    Gabriyelyan, Saak S.; Morris, Sidney A.

    2016-01-01

    We define and study the free topological vector space $\\mathbb{V}(X)$ over a Tychonoff space $X$. We prove that $\\mathbb{V}(X)$ is a $k_\\omega$-space if and only if $X$ is a $k_\\omega$-space. If $X$ is infinite, then $\\mathbb{V}(X)$ contains a closed vector subspace which is topologically isomorphic to $\\mathbb{V}(\\mathbb{N})$. It is proved that if $X$ is a $k$-space, then $\\mathbb{V}(X)$ is locally convex if and only if $X$ is discrete and countable. If $X$ is a metrizable space it is shown ...

  15. Matrix vector analysis

    CERN Document Server

    Eisenman, Richard L

    2005-01-01

    This outstanding text and reference applies matrix ideas to vector methods, using physical ideas to illustrate and motivate mathematical concepts but employing a mathematical continuity of development rather than a physical approach. The author, who taught at the U.S. Air Force Academy, dispenses with the artificial barrier between vectors and matrices--and more generally, between pure and applied mathematics.Motivated examples introduce each idea, with interpretations of physical, algebraic, and geometric contexts, in addition to generalizations to theorems that reflect the essential structur

  16. Scalar-vector bootstrap

    Energy Technology Data Exchange (ETDEWEB)

    Rejon-Barrera, Fernando [Institute for Theoretical Physics, University of Amsterdam,Science Park 904, Postbus 94485, 1090 GL, Amsterdam (Netherlands); Robbins, Daniel [Department of Physics, Texas A& M University,TAMU 4242, College Station, TX 77843 (United States)

    2016-01-22

    We work out all of the details required for implementation of the conformal bootstrap program applied to the four-point function of two scalars and two vectors in an abstract conformal field theory in arbitrary dimension. This includes a review of which tensor structures make appearances, a construction of the projectors onto the required mixed symmetry representations, and a computation of the conformal blocks for all possible operators which can be exchanged. These blocks are presented as differential operators acting upon the previously known scalar conformal blocks. Finally, we set up the bootstrap equations which implement crossing symmetry. Special attention is given to the case of conserved vectors, where several simplifications occur.

  17. Multithreading in vector processors

    Energy Technology Data Exchange (ETDEWEB)

    Evangelinos, Constantinos; Kim, Changhoan; Nair, Ravi

    2018-01-16

    In one embodiment, a system includes a processor having a vector processing mode and a multithreading mode. The processor is configured to operate on one thread per cycle in the multithreading mode. The processor includes a program counter register having a plurality of program counters, and the program counter register is vectorized. Each program counter in the program counter register represents a distinct corresponding thread of a plurality of threads. The processor is configured to execute the plurality of threads by activating the plurality of program counters in a round robin cycle.

  18. Targeted Gene Deletion Using DNA-Free RNA-Guided Cas9 Nuclease Accelerates Adaptation of CHO Cells to Suspension Culture.

    Science.gov (United States)

    Lee, Namil; Shin, JongOh; Park, Jin Hyoung; Lee, Gyun Min; Cho, Suhyung; Cho, Byung-Kwan

    2016-11-18

    Chinese hamster ovary (CHO) cells are the preferred host for the production of a wide array of biopharmaceuticals. Thus, efficient and rational CHO cell line engineering methods have been in high demand to improve quality and productivity. Here, we provide a novel genome engineering platform for increasing desirable phenotypes of CHO cells based upon the integrative protocol of high-throughput RNA sequencing and DNA-free RNA-guided Cas9 (CRISPR associated protein9) nuclease-based genome editing. For commercial production of therapeutic proteins, CHO cells have been adapted for suspension culture in serum-free media, which is highly beneficial with respect to productivity and economics. To engineer CHO cells for rapid adaptation to a suspension culture, we exploited strand-specific RNA-seq to identify genes differentially expressed according to their adaptation trajectory in serum-free media. More than 180 million sequencing reads were generated and mapped to the currently available 109,152 scaffolds of the CHO-K1 genome. We identified significantly downregulated genes according to the adaptation trajectory and then verified their effects using the genome editing method. Growth-based screening and targeted amplicon sequencing revealed that the functional deletions of Igfbp4 and AqpI gene accelerate suspension adaptation of CHO-K1 cells. The availability of this strand-specific transcriptome sequencing and DNA-free RNA-guided Cas9 nuclease mediated genome editing facilitates the rational design of the CHO cell genome for efficient production of high quality therapeutic proteins.

  19. Arsenic trioxide preferentially induces nonapoptotic cell deaths as well as actin cytoskeleton rearrangement in the CHO AA8 cell line

    Directory of Open Access Journals (Sweden)

    Magdalena Izdebska

    2014-12-01

    Full Text Available Introduction: The therapeutic effect of arsenic trioxide (ATO, As2O3 has been investigated for many years. However, the precise molecular mechanisms underlying the antitumor activity of ATO are still not fully understood, but seem to depend on cell types, dosage, and duration of exposure. The purpose of this study was to assess the actin cytoskeleton rearrangement during the cell death process induced by arsenic trioxide in the CHO AA8 cells. A better understanding the mechanisms of ATO-action is likely to lead to more rational use of this drug either as monotherapies or in combination with other anticancer agents.Material and methods: The effect of ATO on actin cytoskeleton was studied in Chinese Hamster Ovary AA8 cell line. Actin was visualized by fluorescence microscopy and phalloidin conjugated to Alexa Fluor® 488. Morphological and ultrastructural alterations in the CHO AA8 cells were evaluated by using light and electron microscope, respectively. For quantitative measurement of cell death, Annexin V-Alexa Fluor® 488 and Propidium Iodide assay was performed. The vital staining of CHO AA8 cells with acridine orange was applied to detect the development of acidic vesicular organelles (AVOs.Results: The performed experiments revealed a dose-dependent decrease in the cell survival. The morphological and ultrastructural features acquired by the cells after ATO-treatment were considered as typical for autophagy and mitotic cell death. As was shown by acridine orange staining, arsenic trioxide treatment increased red fluorescence signals in dose-dependent manner, indicating the development of AVOs, a hallmark of autophagy. Low level of apoptosis was induced in the ATO-treated CHO AA8 cells. Furthermore, the rearrangement of actin filaments associated with cell death process was also detected.Conclusions: The obtained results suggest that arsenic trioxide preferentially induces nonapoptotic cell deaths, autophagy and mitotic cell death, in p53

  20. Determination of IGF-1-Producing CHO-K1 Growth Phases Using GCMS-Based Global Metabolite Analysis

    Directory of Open Access Journals (Sweden)

    S. E. M. SABERI

    2011-12-01

    Full Text Available Mammalian cell lines, in particular CHO-K1 is vital for the multibillion dollar biotechnology industry. The majority of large scale bioprocessing of commercially valuable protein biopharmaceuticals is produced using this type of cell. An ideal mammalian cell system as host for biologics production should retain efficient use of energy sources in order to boost productivity at minimum cost. Various analyses such as cell counting and monitoring of specific biochemical responses are used to provide data to enable bioprocess control in order to achieve the ideal system. Our study aimed to see whether global metabolite analysis using Gas Chromatography Mass Spectrometry (GCMS would be a potential alternative approach in providing data for bioprocess control. In this study, we analyzed metabolites of CHO-K1 cells at different growth phases using GCMS. CHO-K1 cells producing insulin like growth factor-I (IGF1 were obtained from ATCC. Cells were grown in T-flask and incubated at 37°C/ 5% CO2 until 70-80% confluent in RPMI 1640 media. Samples (cells and spent/conditioned media were taken at designated intervals for routine cell counting (Trypan Blue dye exclusion method; glucose, glutamine and lactate determination (YSI 2700; IGF-1 production (ELISA kit R&D Sstems, Inc; and global metabolite analysis (GCMS. Conditioned media from each time point were spun down before subjecting into GCMS. Data from GCMS was then transferred to SIMCA P+12.0 for chemometric evaluation using Principal Component Analysis (PCA. The first component, PC1 results was able to explain 36% of the variation of the data with clear separation between exponential phase and other phases (initial and death phase. This suggests that GCMS-based global metabolite analysis has the ability to capture cell growth behaviour and offered insights of factors that may influence the biological system.ABSTRAK: Produk yang berupa sel kekal mamalia, terutamnya CHO-K1 adalah penting dan menguntungkan

  1. Enhancement of thematic mapper satellite images for geological mapping of the Cho Dien area, Northern Vietnam

    Science.gov (United States)

    Won-In, Krit; Charusiri, Punya

    2003-06-01

    Information available from the earth science image processing package (ESIPP) software program was applied to enhance the satellite image data of the Cho Dien area, northern Vietnam. The area with dense vegetation covers is dominated by several small Zn-Pb prospects in middle Paleozoic limestone units. Interpretation of satellite image data using the digital enhancement ESIPP program, forms the prime objective of this study, which is to improve the image quality and visual interpretation of regional geology, lineament and structural geology. Thematic mapper of bands 7, 5 and 4 with the false-color composites: blue, green and red, respectively, are considered to be the most appropriate for geologic interpretation. Dark pixel correction is carried out prior to other enhancement analyses which include high-pass filtering, albedo correction, image classification, principle component analysis (PCA) and band ratios. High-pass filtering enhancement is considered to be the most suitable approach for lineament analysis. Albedo is good for differentiating lithology, and image classification is also successfully used for lineament interpretation and discrimination of lithologies but is regarded not better than high-pass filtering and albedo. PCA and ratio of band enhancements are considered not good because there are many disturbed and excavated land areas such as abandoned and current open pits in the concerned area. The result of Landsat interpretation indicate that most lineament structures developed in a roughly N-trending anticlinal structure are in NE-, E- and N-trends. Minor lineaments are in roughly NW-trend, and cross-cutting the NE- and E-trends. Interpretation from enhanced Landsat information also fits very well with field evidences. The interpreted map is slightly different from those of the previous mapping works, particularly with respect to detailed lithological boundaries.

  2. Profiling of N-glycosylation gene expression in CHO cell fed-batch cultures.

    Science.gov (United States)

    Wong, Danny Chee Furng; Wong, Niki Soo Ching; Goh, John Soo Yang; May, Lee May; Yap, Miranda Gek Sim

    2010-10-15

    One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N-glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N-glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed-batch culture of a CHO cell line producing recombinant human interferon-gamma (IFN-gamma). Of the 24 N-glycosylation genes examined, 21 showed significant up- or down-regulation of gene expression as the fed-batch culture progressed from exponential, stationary and death phase. As the fed-batch culture progressed, there was also an increase in less sialylated IFN-gamma glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN-gamma. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed-batch strategy appears to need a 0.5 mM glutamine threshold to maintain similar N-glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible "bottlenecks" or "compromised" pathways in N-glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. Copyright 2010 Wiley Periodicals, Inc.

  3. A novel sugar analog enhances sialic acid production and biotherapeutic sialylation in CHO cells.

    Science.gov (United States)

    Yin, Bojiao; Wang, Qiong; Chung, Cheng-Yu; Bhattacharya, Rahul; Ren, Xiaozhi; Tang, Juechun; Yarema, Kevin J; Betenbaugh, Michael J

    2017-08-01

    A desirable feature of many therapeutic glycoprotein production processes is to maximize the final sialic acid content. In this study, the effect of applying a novel chemical analog of the sialic acid precursor N-acetylmannosamine (ManNAc) on the sialic acid content of cellular proteins and a model recombinant glycoprotein, erythropoietin (EPO), was investigated in CHO-K1 cells. By introducing the 1,3,4-O-Bu 3 ManNAc analog at 200-300 µM into cell culture media, the intracellular sialic acid content of EPO-expressing cells increased ∼8-fold over untreated controls while the level of cellular sialylated glycoconjugates increased significantly as well. For example, addition of 200-300 µM 1,3,4-O-Bu 3 ManNAc resulted in >40% increase in final sialic acid content of recombinant EPO, while natural ManNAc at ∼100 times higher concentration of 20 mM produced a less profound change in EPO sialylation. Collectively, these results indicate that butyrate-derivatization of ManNAc improves the capacity of cells to incorporate exogenous ManNAc into the sialic acid biosynthetic pathway and thereby increase sialylation of recombinant EPO and other glycoproteins. This study establishes 1,3,4-O-Bu 3 ManNAc as a novel chemical supplement to improve glycoprotein quality and sialylation levels at concentrations orders of magnitude lower than alternative approaches. Biotechnol. Bioeng. 2017;114: 1899-1902. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  4. Support Vector Components Analysis

    NARCIS (Netherlands)

    van der Ree, Michiel; Roerdink, Johannes; Phillips, Christophe; Garraux, Gaetan; Salmon, Eric; Wiering, Marco

    2017-01-01

    In this paper we propose a novel method for learning a distance metric in the process of training Support Vector Machines (SVMs) with the radial basis function kernel. A transformation matrix is adapted in such a way that the SVM dual objective of a classification problem is optimized. By using a

  5. Sesquilinear uniform vector integral

    Indian Academy of Sciences (India)

    1Faculty of Mathematics and Computer Science, University of Bucharest, Bucharest,. Academiei Str., 14, 010014, Romania. 2Technical University of Civil ... an integral of scalar functions with respect to vector measures, Dunford and his school introduced the spectral operators, thus founding the present operator theory (see ...

  6. Orthogonalisation of Vectors

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 5; Issue 3. Orthogonalisation of Vectors - Matrix Decomposition and Approximation Problems. Rajendra Bhatia. General Article Volume 5 ... Author Affiliations. Rajendra Bhatia1. Indian Statistical Institute 7, SJS Sansanwal Marg, New Delhi 110 016, India.

  7. Calculus with vectors

    CERN Document Server

    Treiman, Jay S

    2014-01-01

    Calculus with Vectors grew out of a strong need for a beginning calculus textbook for undergraduates who intend to pursue careers in STEM. fields. The approach introduces vector-valued functions from the start, emphasizing the connections between one-variable and multi-variable calculus. The text includes early vectors and early transcendentals and includes a rigorous but informal approach to vectors. Examples and focused applications are well presented along with an abundance of motivating exercises. All three-dimensional graphs have rotatable versions included as extra source materials and may be freely downloaded and manipulated with Maple Player; a free Maple Player App is available for the iPad on iTunes. The approaches taken to topics such as the derivation of the derivatives of sine and cosine, the approach to limits, and the use of "tables" of integration have been modified from the standards seen in other textbooks in order to maximize the ease with which students may comprehend the material. Additio...

  8. Vector-borne Infections

    Centers for Disease Control (CDC) Podcasts

    2011-04-18

    This podcast discusses emerging vector-borne pathogens, their role as prominent contributors to emerging infectious diseases, how they're spread, and the ineffectiveness of mosquito control methods.  Created: 4/18/2011 by National Center for Emerging Zoonotic and Infectious Diseases (NCEZID).   Date Released: 4/27/2011.

  9. Over-expressed human TREK-1 inhibits CHO cell proliferation via inhibiting PKA and p38 MAPK pathways and subsequently inducing G1 arrest.

    Science.gov (United States)

    Zhang, Man; Yin, Hua-Jing; Wang, Wei-Ping; Li, Jiang; Wang, Xiao-Liang

    2016-09-01

    Recent studies have shown that the two-pore-domain potassium channel TREK-1 is involved in the proliferation of neural stem cells, astrocytes and human osteoblasts. In this study, we investigated how TREK-1 affected the proliferation of Chinese hamster ovary (CHO) cells in vitro. A CHO cell line stably expressing hTREK-1 (CHO/hTREK-1 cells) was generated. TREK-1 channel currents in the cells were recorded using whole-cell voltage-clamp recording. The cell cycle distribution was assessed using flow cytometry analysis. The expression of major signaling proteins involved was detected with Western blotting. CHO/hTREK-1 cells had a high level of TREK-1 expression, reached up to 320%±16% compared to the control cells. Application of arachidonic acid (10 μmol/L), chloroform (1 mmol/L) or etomidate (10 μmol/L) substantially increased TREK-1 channel currents in CHO/hTREK-1 cells. Overexpression of TREK-1 caused CHO cells arresting at the G1 phase, and significantly decreased the expression of cyclin D1. The TREK-1 inhibitor l-butylphthalide (1-100 μmol/L) dose-dependently attenuated TREK-1-induced G1 phase cell arrest. Moreover, overexpression of TREK-1 significantly decreased the phosphorylation of Akt (S473), glycogen synthase kinase-3β (S9) and cAMP response element-binding protein (CREB, S133), enhanced the phosphorylation of p38 (T180/Y182), but did not alter the phosphorylation and expression of signal transducer and activator of transcription 3 (STAT3). TREK-1 overexpression suppresses CHO cell proliferation by inhibiting the activity of PKA and p38/MAPK signaling pathways and subsequently inducing G1 phase cell arrest.

  10. Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts.

    Directory of Open Access Journals (Sweden)

    Ana de la Cueva

    Full Text Available Colorectal cancer (CRC is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα, an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy.ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS and thymidine kinase (TK1 levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action.Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

  11. Helices and vector bundles

    CERN Document Server

    Rudakov, A N

    1990-01-01

    This volume is devoted to the use of helices as a method for studying exceptional vector bundles, an important and natural concept in algebraic geometry. The work arises out of a series of seminars organised in Moscow by A. N. Rudakov. The first article sets up the general machinery, and later ones explore its use in various contexts. As to be expected, the approach is concrete; the theory is considered for quadrics, ruled surfaces, K3 surfaces and P3(C).

  12. Anisotropic vector Preisach particle

    CERN Document Server

    Fuezi, J

    2000-01-01

    The static 2D vector magnetic behaviour of an anisotropic silicon iron sheet is modelled by a particle which depicts its space-averaged behaviour. The magnitude of magnetization is governed by a classical Preisach operator with the projection of field strength on the magnetization direction as input. Its orientation is determined by the equilibrium between the field strength orientation and the anisotropy of the sheet.

  13. What is a vector?

    Science.gov (United States)

    Morgan, Eric René; Booth, Mark; Norman, Rachel; Mideo, Nicole; McCallum, Hamish; Fenton, Andy

    2017-01-01

    Many important and rapidly emerging pathogens of humans, livestock and wildlife are ‘vector-borne’. However, the term ‘vector’ has been applied to diverse agents in a broad range of epidemiological systems. In this perspective, we briefly review some common definitions, identify the strengths and weaknesses of each and consider the functional differences between vectors and other hosts from a range of ecological, evolutionary and public health perspectives. We then consider how the use of designations can afford insights into our understanding of epidemiological and evolutionary processes that are not otherwise apparent. We conclude that from a medical and veterinary perspective, a combination of the ‘haematophagous arthropod’ and ‘mobility’ definitions is most useful because it offers important insights into contact structure and control and emphasizes the opportunities for pathogen shifts among taxonomically similar species with similar feeding modes and internal environments. From a population dynamics and evolutionary perspective, we suggest that a combination of the ‘micropredator’ and ‘sequential’ definition is most appropriate because it captures the key aspects of transmission biology and fitness consequences for the pathogen and vector itself. However, we explicitly recognize that the value of a definition always depends on the research question under study. This article is part of the themed issue ‘Opening the black box: re-examining the ecology and evolution of parasite transmission’. PMID:28289253

  14. Lentiviral Vector Mediated Transgenesis.

    Science.gov (United States)

    Barde, Isabelle; Verp, Sonia; Offner, Sandra; Trono, Didier

    2011-03-01

    The genetic manipulation of rodents through the generation of fully transgenic animals or via the modification of selective cells or organs is a procedure of paramount importance for biomedical research, either to address fundamental questions or to develop preclinical models of human diseases. Lentiviral vectors occupy the front stage in this scene, as they can mediate the integration and stable expression of transgenes both in vitro and in vivo. Widely used to modify a variety of cells, including re-implantable somatic and embryonic stem cells, lentiviral vectors can also be directly administered in vivo, for instance in the brain. However, perhaps their most spectacular research application is in the generation of transgenic animals. Compared with the three-decade-old DNA pronuclear injection technique, lentivector-mediated transgenesis is simple, cheap, and highly efficient. Furthermore, it can take full advantage of the great diversity of lentiviral vectors developed for other applications, and thus allows for ubiquitous or tissue-specific or constitutive or externally controllable transgene expression, as well as RNAi-mediated gene knockdown. Curr. Protoc. Mouse Biol. 1:169-184. © 2011 by John Wiley & Sons, Inc. Copyright © 2011 John Wiley & Sons, Inc.

  15. Propagating Gateway Vectors.

    Science.gov (United States)

    Reece-Hoyes, John S; Walhout, Albertha J M

    2018-01-02

    Generating stocks of Entry and Destination vectors for use in the Gateway recombinatorial cloning system requires transforming them into Escherichia coli strain DB3.1, where they can replicate because this strain is immune to the effects of the ccdB gene carried in the Gateway cassette. However, mutations in the ccdB gene can arise at low frequency, and these mutant plasmids will consequently allow growth of standard cloning strains of E. coli (e.g., DH5α). Therefore, after making new stocks of Gateway plasmids, their ability to grow in cloning strains of E. coli must be tested. This involves obtaining multiple stocks of vector, each arising from a single plasmid grown in a single DB3.1 bacterial colony, and transforming each stock into both DB3.1 and the preferred cloning strain of E. coli in a controlled fashion. Only vector stocks that effectively kill the standard cloning strain (i.e., no or few colonies are obtained after transformation) should be used in Gateway cloning reactions. The sequence can be performed in 3 d. © 2018 Cold Spring Harbor Laboratory Press.

  16. Protective effect of propolis on radiation-induced chromosomal damage on Chinese hamster ovary cells (CHO-K1)

    Energy Technology Data Exchange (ETDEWEB)

    Spigoti, Geyza; Bartolini, Paolo; Okazaki, Kayo [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)], e-mail: kokazaki@ipen.br; Tsutsumi, Shiguetoshi [Amazon Food Ltd., Tokyo (Japan)], e-mail: fwip5138@mb.infoweb.ne.jp

    2009-07-01

    In the last years, particular interest has been given to investigations concerning natural, effective and nontoxic compounds with radioprotective capacity in concert with increasing utilization of different types of ionizing radiation for various applications. Among them, propolis, a resinous mixture of substances collected by honey bees (Apis mellifera) has been considered promising since it presents several advantageous characteristics, i.e., antiinflammatory, anticarcinogenic, antimicrobial and free radical scavenging action. It is, therefore, a direct antioxidant that protects cells and organisms from the adverse effects of ionizing radiation. These relevant biological activities are mainly mediated by the flavonoids, present at relatively high concentrations in the propolis. Considering that the chemical composition and, consequently, the biological activity of propolis is variable according to the environmental plant ecology, the present study was conducted in order to evaluate the radioprotective capacity of Brazilian propolis, collected in the State of Rio Grande do Sul, against genotoxic damages induced by {sup 60}Co {gamma}-radiation in Chinese hamster ovary cells (CHO-K1). for this purpose, micronucleus induction was analyzed concerning irreparable damage, specifically related to DNA double-strand breaks, that are potentially carcinogenic. CHO-K1 cells were submitted to different concentrations of propolis (3 - 33 {mu}g/ml), 1 h before irradiation, with 1 Gy of {gamma} radiation (0.722 Gy/min). The data obtained showed a decreasing tendency in the quantity of radioinduced damage on cells previously treated with propolis. The radioprotective effect was more prominent at higher propolis concentration. The treatment with propolis alone did not induce genotoxic effects on CHO-K1 cells. Beside that, the treatment with propolis, associated or not with radiation, did not influence the kinetics of cellular proliferation. (author)

  17. Cytotoxicity Evaluation of Anatase and Rutile TiO2 Thin Films on CHO-K1 Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Blanca Cervantes

    2016-07-01

    Full Text Available Cytotoxicity of titanium dioxide (TiO2 thin films on Chinese hamster ovary (CHO-K1 cells was evaluated after 24, 48 and 72 h of culture. The TiO2 thin films were deposited using direct current magnetron sputtering. These films were post-deposition annealed at different temperatures (300, 500 and 800 °C toward the anatase to rutile phase transformation. The root-mean-square (RMS surface roughness of TiO2 films went from 2.8 to 8.08 nm when the annealing temperature was increased from 300 to 800 °C. Field emission scanning electron microscopy (FESEM results showed that the TiO2 films’ thickness values fell within the nanometer range (290–310 nm. Based on the results of the tetrazolium dye and trypan blue assays, we found that TiO2 thin films showed no cytotoxicity after the aforementioned culture times at which cell viability was greater than 98%. Independently of the annealing temperature of the TiO2 thin films, the number of CHO-K1 cells on the control substrate and on all TiO2 thin films was greater after 48 or 72 h than it was after 24 h; the highest cell survival rate was observed in TiO2 films annealed at 800 °C. These results indicate that TiO2 thin films do not affect mitochondrial function and proliferation of CHO-K1 cells, and back up the use of TiO2 thin films in biomedical science.

  18. Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media.

    Science.gov (United States)

    Miki, Hideo; Takagi, Mutsumi

    2015-08-01

    The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.

  19. Cytotoxicity and genotoxicity of stilbene derivatives in CHO-K1 and HepG2 cell lines

    Directory of Open Access Journals (Sweden)

    Cassia Suemi Mizuno

    2017-07-01

    Full Text Available Abstract The cytotoxicity and genotoxicity of the stilbenes (E-methyl-4-(3-5-dimethoxystyrylbenzoate (ester, (E-4-(3-5-dimethoxystyrylaniline (amino, (Z-1,3-dimethoxy-5-(4-methoxystyrylbenzene (cis-TMS and (E-1,3-dimethoxy-5-(4-methoxystyrylbenzene (trans-TMS were investigated in this work. Structural modifications of resveratrol, a naturally occurring stilbene, have been previously performed, including the replacement of hydroxyl by different functional groups. Such modifications resulted in significant improvement of target-specific effects on cell death and antiproliferative responses. The parameters were evaluated using XTT assay, clonogenic survival assay and the cytokinesis-block micronucleus assay in CHO-K1 and HepG2 cell lines. The results showed that cis-TMS is approximately 250-fold more cytotoxic than the amino and ester, and 128-fold more cytotoxic than trans-TMS. When genotoxicity was evaluated, only the trans-TMS did not significantly increase the frequency of micronucleus (MN. While the cis-TMS induced a mean of 5.2 and 5.9 MN/100 cells at 0.5 μM in CHO-K1 and HepG2, respectively, the amino and ester induced 3.1 and 3.6 MN/100 cells at 10 μM in CHO-K1, respectively, and 3.5 and 3.8 in HepG2. Trans-TMS is genotoxic only in HepG2 cells. Based on these results, the cis-TMS was the most cytotoxic and genotoxic compound in both cell lines.

  20. Brønsted acid catalyzed enantioselective indole aza-Claisen rearrangement mediated by an arene CH-O interaction.

    Science.gov (United States)

    Maity, Pradip; Pemberton, Ryan P; Tantillo, Dean J; Tambar, Uttam K

    2013-11-06

    Although the aromatic aza-Claisen rearrangement is a general strategy for accessing substituted aromatic amines, there are no highly enantioselective examples of this process. We report the first Brønsted acid catalyzed enantioselective indole aza-Claisen rearrangement for the synthesis of chiral 3-amino-2-substituted indoles. We present evidence for an arene CH-O interaction as a source of activation and stereoinduction, which is an unprecedented phenomenon in enantioselective Brønsted acid catalysis. The products of this reaction can be transformed into 3-aminooxindoles, which are prevalent in many biologically active small molecules.

  1. DNA double-strand break induction in Ku80-deficient CHO cells following Boron Neutron Capture Reaction

    Directory of Open Access Journals (Sweden)

    Masunaga Shinichiro

    2011-09-01

    Full Text Available Abstract Background Boron neutron capture reaction (BNCR is based on irradiation of tumors after accumulation of boron compound. 10B captures neutrons and produces an alpha (4He particle and a recoiled lithium nucleus (7Li. These particles have the characteristics of high linear energy transfer (LET radiation and have marked biological effects. The purpose of this study is to verify that BNCR will increase cell killing and slow disappearance of repair protein-related foci to a greater extent in DNA repair-deficient cells than in wild-type cells. Methods Chinese hamster ovary (CHO-K1 cells and a DNA double-strand break (DSB repair deficient mutant derivative, xrs-5 (Ku80 deficient CHO mutant cells, were irradiated by thermal neutrons. The quantity of DNA-DSBs following BNCR was evaluated by measuring the phosphorylation of histone protein H2AX (gamma-H2AX and 53BP1 foci using immunofluorescence intensity. Results Two hours after neutron irradiation, the number of gamma-H2AX and 53BP1 foci in the CHO-K1 cells was decreased to 36.5-42.8% of the levels seen 30 min after irradiation. In contrast, two hours after irradiation, foci levels in the xrs-5 cells were 58.4-69.5% of those observed 30 min after irradiation. The number of gamma-H2AX foci in xrs-5 cells at 60-120 min after BNCT correlated with the cell killing effect of BNCR. However, in CHO-K1 cells, the RBE (relative biological effectiveness estimated by the number of foci following BNCR was increased depending on the repair time and was not always correlated with the RBE of cytotoxicity. Conclusion Mutant xrs-5 cells show extreme sensitivity to ionizing radiation, because xrs-5 cells lack functional Ku-protein. Our results suggest that the DNA-DSBs induced by BNCR were not well repaired in the Ku80 deficient cells. The RBE following BNCR of radio-sensitive mutant cells was not increased but was lower than that of radio-resistant cells. These results suggest that gamma-ray resistant cells have

  2. Construction of an expression vector for propionibacteria and its use in production of 5-aminolevulinic acid by Propionibacterium freudenreichii.

    Science.gov (United States)

    Kiatpapan, P; Murooka, Y

    2001-07-01

    Several promoters from Propionibacterium freudenreichii subsp. shermanii were isolated using a promoter probe vector, pCVE1, containing the Streptomyces cholesterol oxidase gene (choA) as a reporter gene. Three of four promoters isolated exhibiting a strong activity in Escherichia coli also expressed a strong activity in P. freudenreichii subsp. shermanii IFO12426. Using two promoters with a strong activity and a previously constructed shuttle vector, pPK705, shuttling between E. coli and Propionibacterium. we constructed expression vectors for propionibacteria. To overproduce 5-aminolevulinic acid (ALA), which is the first intermediate in the synthesis of porphyrins, the ALA synthase gene (hemA) from Rhodobacter sphaeroides was recombined with the expression vectors. The activity of ALA synthase in the recombinant P freudenreichii subsp. shermanii increased about 70-fold that in the strain without a vector. The recombinant Propionibacterium produced ALA at a maximum concentration of 8.6 mM in the absence of levulinic acid, an inhibitor of ALA dehydratase, with 1% glucose as a carbon source. The recombinant P. freudenreichii accumulated 18.8 mmol/g cells ALA in the presence of 1 mM levulinic acid and 30 mM glycine. The construction of an efficient expression vector will facilitate genetic studies of a vitamin B12 producer, Propionibacterium.

  3. Stable expression of human thyrotropin (hTSH) in mammalian cells (CHO) expressing {alpha}2,6 sialyltransferase; Expressao estavel tireotrofina humana (r-hTSH) em celulas de mamifero (CHO) que expressam {alpha}2,6 sialiltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Damiani, Renata

    2009-07-01

    A CHO cell line, previously genetically modified by the introduction of rat {alpha}2,6-sialyltransferase cDNA, generated for the first time a human-like sialylated recombinant hTSH (hlsr-hTSH) more similar to the native hormone, with 61% of {alpha}2,3- and 39% of {alpha}2,6-linked sialic acid residues. The best clone, when submitted to gene amplification with up to 8 {mu}M methotrexate, presented a secretion level of {approx}2 {mu}g hTSH/10{sup 6} cells/day, useful for product purification and characterization. The relative molecular masses (M{sub r}) of the heterodimer and of the {alpha}- and {beta}-subunits of purified hlsr-hTSH, determined by MALDI-TOF mass spectrometry, and the relative hydrophobicities, determined by RP-HPLC, were not remarkably different from those presented by two r-hTSH preparations secreted by normal CHO cells. Some differences were observed, though, in N-glycan composition, with more tri- and much more tetra-sialylated structures in hlsr-hTSH. When analyzed via an in vivo bioassay based on hTSH-induced T{sub 4} release in mice, hlsr-hTSH was shown to be equipotent (p > 0.05) with the commercial preparation of r-hTSH (Thyrogen), and 1.5-fold more potent than native hTSH (p < 0.001). (author)

  4. Scalar - vector soliton fiber lasers

    CERN Document Server

    Wu, Zhichao; Li, Lei; Luo, Yiyang; Tang, Dingyuan; Shen, Deyuan; Tang, Ming; Fu, Songnian; Zhao, Luming

    2016-01-01

    Rapid progress in passively mode-locked fiber lasers is currently driven by the recent discovery of vector feature of mode-locking pulses, namely, the group velocity-locked vector solitons, the phase locked vector solitons, and the high-order vector solitons. Those vector solitons are fundamentally different from the previously known scalar solitons. Here, we report a fiber laser where the mode-locked pulse evolves as a vector soliton in the strong birefringent segment and is transformed into a regular scalar soliton after the polarizer within the laser cavity. The existence of solutions in a polarization-dependent cavity comprising a periodic combination of two distinct nonlinear waves is novel and likely to be applicable to various other nonlinear systems. For very large local birefringence, our laser approaches the working regime of vector soliton lasers, while it approaches scalar soliton fiber lasers under the conditions of very small birefringence.

  5. Efficient production of recombinant adeno-associated viral vector, serotype DJ/8, carrying the GFP gene.

    Science.gov (United States)

    Hashimoto, Haruo; Mizushima, Tomoko; Chijiwa, Tsuyoshi; Nakamura, Masato; Suemizu, Hiroshi

    2017-06-15

    The purpose of this study was to establish an efficient method for the preparation of an adeno-associated viral (AAV), serotype DJ/8, carrying the GFP gene (AAV-DJ/8-GFP). We compared the yields of AAV-DJ/8 vector, which were produced by three different combination methods, consisting of two plasmid DNA transfection methods (lipofectamine and calcium phosphate co-precipitation; CaPi) and two virus DNA purification methods (iodixanol and cesium chloride; CsCl). The results showed that the highest yield of AAV-DJ/8-GFP vector was accomplished with the combination method of lipofectamine transfection and iodixanol purification. The viral protein expression levels and the transduction efficacy in HEK293 and CHO cells were not different among four different combination methods for AAV-DJ/8-GFP vectors. We confirmed that the AAV-DJ/8-GFP vector could transduce to human and murine hepatocyte-derived cell lines. These results show that AAV-DJ/8-GFP, purified by the combination of lipofectamine and iodixanol, produces an efficient yield without altering the characteristics of protein expression and AAV gene transduction. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo

    Science.gov (United States)

    Jenke, Bok Hee C.; Fetzer, Christian P.; Stehle, Isa M.; Jönsson, Franziska; Fackelmayer, Frank O.; Conradt, Harald; Bode, Jürgen; Lipps, Hans J.

    2002-01-01

    pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA–protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class. PMID:11897664

  7. Scalar and vector Galileons

    Science.gov (United States)

    Rodríguez, Yeinzon; Navarro, Andrés A.

    2017-03-01

    An alternative for the construction of fundamental theories is the introduction of Galileons. These are fields whose action leads to non higher than second-order equations of motion. As this is a necessary but not sufficient condition to make the Hamiltonian bounded from below, as long as the action is not degenerate, the Galileon construction is a way to avoid pathologies both at the classical and quantum levels. Galileon actions are, therefore, of great interest in many branches of physics, specially in high energy physics and cosmology. This proceedings contribution presents the generalities of the construction of both scalar and vector Galileons following two different but complimentary routes.

  8. Architecture and Vector Control

    DEFF Research Database (Denmark)

    von Seidlein, Lorenz; Knols, Bart GJ; Kirby, Matthew

    2012-01-01

    of vector-borne diseases have no access to electricity. Many houses in the hot, humid regions of Asia have adapted to the environment, they are built of porous materials and are elevated on stilts features which allow a comfortable climate even in the presence of bednets and screens. In contrast, many...... buildings in Africa and Asia in respect to their indoor climate characteristics and finally, show how state-of-the-art 3D modelling can predict climate characteristics and help to optimize buildings....

  9. Women in church and society: Report of research done by a research team at the PU vir CHO

    Directory of Open Access Journals (Sweden)

    F.J. van Rensburg

    2002-08-01

    Full Text Available The research project “Women in Church and Society” was conducted under the auspices of one of the focus areas for research and postgraduate education at the Potchefstroomse Universiteit vir Christelike Hoër Onderwys: “Reformed Theology and the Development of the South African Society”. This focus area is based in the Faculty of Theology (PU vir CHO and is directed by Herrie van Rooy. Project 2 of this focus area is “The socio-historic context of the Bible and its implications for the development of South African Society” and is under the leadership of Fika J. van Rensburg. The first sub-project of Project 2 to be completed is “Women in Church and Society”. It commenced in 2000 and had its fourth and final workshop in September 2002. It was managed by a five-person executive committee and had the following categories of collaborators: 16 PU vir CHO researchers, 10 researchers from other South African universities, 6 international researchers, 19 masters’ and doctoral students, and 21 researchers with special expertise in relevant areas. In total 48 papers1 were read and discussed at the four workshops; and most of them have either been published or are in the process of being published as articles in accredited journals. This article is a report on the activities and outcome of the research project.

  10. Combinatorial treatment with lithium chloride enhances recombinant antibody production in transiently transfected CHO and HEK293E cells

    DEFF Research Database (Denmark)

    Kim, Che Lin; Kwang Ha, Tae; Min Lee, Gyun

    2016-01-01

    Lithium chloride (LiCl), which induces cell cycle arrest at G2/M phase, is known as a specific production rate (qp)-enhancing additive in recombinant Chinese hamster ovary (CHO) cell culture. To determine the potential of LiCl as a chemical additive that enhances transient gene expression (TGE), Li...... of monoclonal antibody (mAb) in CHO-NK cells, pretreatment alone with 10 mM LiCl and post-treatment alone with 5 mM LiCl resulted in 1.2- and 3.4-fold increase of maximum mAb concentration (MMC), respectively, compared with the TGE without LiCl treatment. Furthermore, combinatorial treatment with LiCl (10 m......M for pre-treatment and 5 mM for post-treatment) synergistically increased the TGE of mAb (5.3-fold increase in MMC). Likewise, combinatorial treatment with LiCl (10 mM for pre-treatment and 15 mM for post-treatment) in HEK293E cells synergistically increased the TGE of mAb (4.9-fold increase in MMC). Taken...

  11. Glycoprofiling effects of media additives on IgG produced by CHO cells in fed-batch bioreactors.

    Science.gov (United States)

    Kildegaard, Helene Faustrup; Fan, Yuzhou; Sen, Jette W; Larsen, Bo; Andersen, Mikael R

    2016-02-01

    Therapeutic monoclonal antibodies (mAbs) are mainly produced by heterologous expression in Chinese hamster ovary (CHO) cells. The glycosylation profile of the mAbs has major impact on the efficacy and safety of the drug and is therefore an important parameter to control during production. In this study, the effect on IgG N-glycosylation from feeding CHO cells with eight glycosylation precursors during cultivation was investigated. The study was conducted in fed-batch mode in bioreactors with biological replicates to obtain highly controlled and comparable conditions. We assessed charge heterogeneity and glycosylation patterns of IgG. None of the eight feed additives caused statistically significant changes to cell growth or IgG productivity, compared to controls. However, the addition of 20 mM galactose did result in a reproducible increase of galactosylated IgG from 14% to 25%. On the other hand, addition of 20 mM N-acetyl-D-glucosamine (GlcNAc) reduced relative abundance of galactosylated IgG by 4%. Additionally, supplementation with 10 mM mannose slightly reduced GlcNAc occupancy of IgG. Overall, comparing the effects of IgG glycosylation, by supplementing the cell culture medium with glycosylation precursors during cultivation, revealed an application of these glycosylation precursors for modulating N-glycosylation of IgG. © 2015 Wiley Periodicals, Inc.

  12. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

    Directory of Open Access Journals (Sweden)

    Ryan Haryadi

    Full Text Available Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig heavy chain (HC and kappa light chain (LC was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

  13. Compartment-specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol.

    Science.gov (United States)

    Matuszczyk, Jens-Christoph; Teleki, Attila; Pfizenmaier, Jennifer; Takors, Ralf

    2015-10-01

    Mammalian cells show a compartmented metabolism. Getting access to subcellular metabolite pools is of high interest to understand the cells' metabolomic state. Therefore a protocol is developed and applied for monitoring compartment-specific metabolite and nucleotide pool sizes in Chinese hamster ovary (CHO) cells. The approach consists of a subtracting filtering method separating cytosolic components from physically intact mitochondrial compartments. The internal standards glucose-6-phosphate and cis-aconitate were chosen to quantify cytosolic secession and mitochondrial membrane integrity. Extracts of related fractions were studied by liquid chromatography-isotope dilution mass spectrometry for the absolute quantification of a subset of glycolytic and tricarboxylic acid cycle intermediates together with the adenylate nucleotides ATP, ADP and AMP. The application of the protocol revealed highly dynamic changes in the related pool sizes as a function of distinct cultivation periods of IgG1 producing CHO cells. Mitochondrial and cytosolic pool dynamics were in agreement with anticipated metabolite pools of independent studies. The analysis of adenosine phosphate levels unraveled significantly higher ATP levels in the cytosol leading to the hypothesis that mitochondria predominantly serve for fueling ATP into the cytosol where it is tightly controlled at physiological adenylate energy charges about 0.9. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Co-overexpression of Mgat1 and Mgat4 in CHO cells for production of highly sialylated albumin-erythropoietin.

    Science.gov (United States)

    Cha, Hyun-Myoung; Lim, Jin-Hyuk; Yeon, Jung-Heum; Hwang, Jeong-Min; Kim, Dong-Il

    2017-08-01

    Terminal sialic acids on N-glycan of recombinant human erythropoietin are very important for in vivo half-life, as this glycoprotein has three N-glycosylation sites. N-acetylglucosaminyltransferases I, II, IV, and V (i.e. Mgat1, Mgat2, Mgat4, and Mgat5) catalyze the formation of a glycan antennary structure. These enzymes display different reaction kinetics for a common substrate and generally show low expression in Chinese hamster ovary (CHO) cells. Therefore, genetic control of Mgat expression is an effective method to increase sialic acid contents by enhancing glycan antennarity. To produce highly sialylated albumin-erythropoietin (Alb-EPO), we co-overexpressed the Mgat1 and Mgat4 genes in CHO cells and determined the optimal ratio of Mgat1:Mgat4 gene expression. All transfected cell lines showed increased gene expression of Mgat4, including Mgat1 overexpressing cell line. Sialic acid content of Alb-EPO was highest in co-transfected cells with excess Mgat4 gene, and these cells showed a higher tri- and tetra-antennary structure than control cells. Based on these results, we suggest that co-transfection of the Mgat1 and Mgat4 genes at a ratio of 2:8 is optimal for extension of antennary structures. Also, regulation of Mgat gene expression in the glycan biosynthesis pathway can be a novel approach to increase the terminal sialic acids of N-glycans. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Vector wave propagation method.

    Science.gov (United States)

    Fertig, M; Brenner, K-H

    2010-04-01

    In this paper, we extend the scalar wave propagation method (WPM) to vector fields. The WPM [Appl. Opt.32, 4984 (1993)] was introduced in order to overcome the major limitations of the beam propagation method (BPM). With the WPM, the range of application can be extended from the simulation of waveguides to simulation of other optical elements like lenses, prisms and gratings. In that reference it was demonstrated that the wave propagation scheme provides valid results for propagation angles up to 85 degrees and that it is not limited to small index variations in the axis of propagation. Here, we extend the WPM to three-dimensional vectorial fields (VWPMs) by considering the polarization dependent Fresnel coefficients for transmission in each propagation step. The continuity of the electric field is maintained in all three dimensions by an enhanced propagation vector and the transfer matrix. We verify the validity of the method by transmission through a prism and by comparison with the focal distribution from vectorial Debye theory. Furthermore, a two-dimensional grating is simulated and compared with the results from three-dimensional RCWA. Especially for 3D problems, the runtime of the VWPM exhibits special advantage over the RCWA.

  16. "Analytical" vector-functions I

    Science.gov (United States)

    Todorov, Vladimir Todorov

    2017-12-01

    In this note we try to give a new (or different) approach to the investigation of analytical vector functions. More precisely a notion of a power xn; n ∈ ℕ+ of a vector x ∈ ℝ3 is introduced which allows to define an "analytical" function f : ℝ3 → ℝ3. Let furthermore f (ξ )= ∑n =0 ∞ anξn be an analytical function of the real variable ξ. Here we replace the power ξn of the number ξ with the power of a vector x ∈ ℝ3 to obtain a vector "power series" f (x )= ∑n =0 ∞ anxn . We research some properties of the vector series as well as some applications of this idea. Note that an "analytical" vector function does not depend of any basis, which may be used in research into some problems in physics.

  17. An XBP-1 dependent bottle-neck in production of IgG subtype antibodies in chemically defined serum-free Chinese hamster ovary (CHO) fed-batch processes.

    Science.gov (United States)

    Becker, Eric; Florin, Lore; Pfizenmaier, Klaus; Kaufmann, Hitto

    2008-06-01

    The optimization of production processes for therapeutic antibodies is a continuing challenge in pharmaceutical biotechnology. Although it could be demonstrated that vector design and host cell engineering can improve transcriptional and translational efficiency and thereby result in generation of high producer cell lines, it is not clear whether introduction of transgenes that regulate protein transport or affect post-translational modifications could further improve such industrial processes. Here, we show that heterologous expression of the transcription factor X-box binding protein-1 (XBP-1) can lead to an increase in endoplasmic reticulum (ER) content and specific therapeutic antibody productivity of Chinese hamster ovary (CHO)-DG44 cells in inoculum suspension cultures. This effect translates into 40% increased overall antibody titers in a fed-batch format where cells are grown in chemically defined serum-free media. Protein-A purified antibody products from mock-transfected cells and XBP-1 transfected cells were found to be of comparable quality with regard to glycosylation pattern and physicochemical characteristics. The data demonstrate the potential of XBP-1 engineering to improve mammalian cell culture production processes to yield high amounts of a therapeutic protein product of desired quality.

  18. Optimality Conditions in Vector Optimization

    CERN Document Server

    Jiménez, Manuel Arana; Lizana, Antonio Rufián

    2011-01-01

    Vector optimization is continuously needed in several science fields, particularly in economy, business, engineering, physics and mathematics. The evolution of these fields depends, in part, on the improvements in vector optimization in mathematical programming. The aim of this Ebook is to present the latest developments in vector optimization. The contributions have been written by some of the most eminent researchers in this field of mathematical programming. The Ebook is considered essential for researchers and students in this field.

  19. Rapid transient production in plants by replicating and non-replicating vectors yields high quality functional anti-HIV antibody.

    Science.gov (United States)

    Sainsbury, Frank; Sack, Markus; Stadlmann, Johannes; Quendler, Heribert; Fischer, Rainer; Lomonossoff, George P

    2010-11-12

    The capacity of plants and plant cells to produce large amounts of recombinant protein has been well established. Due to advantages in terms of speed and yield, attention has recently turned towards the use of transient expression systems, including viral vectors, to produce proteins of pharmaceutical interest in plants. However, the effects of such high level expression from viral vectors and concomitant effects on host cells may affect the quality of the recombinant product. To assess the quality of antibodies transiently expressed to high levels in plants, we have expressed and characterised the human anti-HIV monoclonal antibody, 2G12, using both replicating and non-replicating systems based on deleted versions of Cowpea mosaic virus (CPMV) RNA-2. The highest yield (approximately 100 mg/kg wet weight leaf tissue) of affinity purified 2G12 was obtained when the non-replicating CPMV-HT system was used and the antibody was retained in the endoplasmic reticulum (ER). Glycan analysis by mass-spectrometry showed that the glycosylation pattern was determined exclusively by whether the antibody was retained in the ER and did not depend on whether a replicating or non-replicating system was used. Characterisation of the binding and neutralisation properties of all the purified 2G12 variants from plants showed that these were generally similar to those of the Chinese hamster ovary (CHO) cell-produced 2G12. Overall, the results demonstrate that replicating and non-replicating CPMV-based vectors are able to direct the production of a recombinant IgG similar in activity to the CHO-produced control. Thus, a complex recombinant protein was produced with no apparent effect on its biochemical properties using either high-level expression or viral replication. The speed with which a recombinant pharmaceutical with excellent biochemical characteristics can be produced transiently in plants makes CPMV-based expression vectors an attractive option for biopharmaceutical development

  20. Rapid transient production in plants by replicating and non-replicating vectors yields high quality functional anti-HIV antibody.

    Directory of Open Access Journals (Sweden)

    Frank Sainsbury

    2010-11-01

    Full Text Available The capacity of plants and plant cells to produce large amounts of recombinant protein has been well established. Due to advantages in terms of speed and yield, attention has recently turned towards the use of transient expression systems, including viral vectors, to produce proteins of pharmaceutical interest in plants. However, the effects of such high level expression from viral vectors and concomitant effects on host cells may affect the quality of the recombinant product.To assess the quality of antibodies transiently expressed to high levels in plants, we have expressed and characterised the human anti-HIV monoclonal antibody, 2G12, using both replicating and non-replicating systems based on deleted versions of Cowpea mosaic virus (CPMV RNA-2. The highest yield (approximately 100 mg/kg wet weight leaf tissue of affinity purified 2G12 was obtained when the non-replicating CPMV-HT system was used and the antibody was retained in the endoplasmic reticulum (ER. Glycan analysis by mass-spectrometry showed that the glycosylation pattern was determined exclusively by whether the antibody was retained in the ER and did not depend on whether a replicating or non-replicating system was used. Characterisation of the binding and neutralisation properties of all the purified 2G12 variants from plants showed that these were generally similar to those of the Chinese hamster ovary (CHO cell-produced 2G12.Overall, the results demonstrate that replicating and non-replicating CPMV-based vectors are able to direct the production of a recombinant IgG similar in activity to the CHO-produced control. Thus, a complex recombinant protein was produced with no apparent effect on its biochemical properties using either high-level expression or viral replication. The speed with which a recombinant pharmaceutical with excellent biochemical characteristics can be produced transiently in plants makes CPMV-based expression vectors an attractive option for

  1. Estimation of vector velocity

    DEFF Research Database (Denmark)

    2000-01-01

    Using a pulsed ultrasound field, the two-dimensional velocity vector can be determined with the invention. The method uses a transversally modulated ultrasound field for probing the moving medium under investigation. A modified autocorrelation approach is used in the velocity estimation. The new...... estimator automatically compensates for the axial velocity, when determining the transverse velocity by using fourth order moments rather than second order moments. The estimation is optimized by using a lag different from one in the estimation process, and noise artifacts are reduced by using averaging...... of RF samples. Further, compensation for the axial velocity can be introduced, and the velocity estimation is done at a fixed depth in tissue to reduce spatial velocity dispersion....

  2. Multistage vector (MSV) therapeutics

    Science.gov (United States)

    Wolfram, Joy; Shen, Haifa; Ferrari, Mauro

    2015-01-01

    One of the greatest challenges in the field of medicine is obtaining controlled distribution of systemically administered therapeutic agents within the body. Indeed, biological barriers such as physical compartmentalization, pressure gradients, and excretion pathways adversely affect localized delivery of drugs to pathological tissue. The diverse nature of these barriers requires the use of multifunctional drug delivery vehicles that can overcome a wide range of sequential obstacles. In this review, we explore the role of multifunctionality in nanomedicine by primarily focusing on multistage vectors (MSVs). The MSV is an example of a promising therapeutic platform that incorporates several components, including a microparticle, nanoparticles, and small molecules. In particular, these components are activated in a sequential manner in order to successively address transport barriers. PMID:26264836

  3. Interactive effects of zearalenone and its metabolites on cytotoxicity and metabolization in ovarian CHO-K1 cells.

    Science.gov (United States)

    Tatay, Elena; Meca, Giuseppe; Font, Guillermina; Ruiz, Maria-Jose

    2014-02-01

    Zearalenone (ZEA) is a non-steroidal estrogen mycotoxin with high binding affinity to estrogen receptors. ZEA is rapidly absorbed and metabolized in vivo to α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL). So, mixtures of them may be present in biological systems and suppose a hazard to animals and human health. The aims of this study were to determine the cytotoxic effects of ZEA and its metabolites, alone and in combination in ovarian (CHO-K1) cells during 24, 48 and 72h by the MTT assay; and to investigate the metabolism of the CHO-K1 cells on ZEA, and its conversion into α-ZOL and β-ZOL by CHO-K1 cell after 24 and 48h of exposure. The IC50 value obtained for individual mycotoxins range from 60.3 to >100.0μM, from 30.0 to 33.0μM and from 55.0 to >75.0μM for ZEA, α-ZOL and β-ZOL, respectively. Cytotoxic interactions were assayed by the isobologram method, which provides a combination index (CI) value as a quantitative measure of the degree of the three mycotoxin interaction. The CI values for binary combinations ranged from 0.56±0.15 (synergism at low concentrations) to 5.25±5.10 (addition at high concentrations) and tertiary combinations from 2.95±0.75 (antagonism at low concentrations) to 0.41±0.23 (synergism at high concentrations). The concentration of ZEA and its metabolites was determined with liquid chromatography coupled to the mass spectrometer detector-linear ion trap (LC-MS-LIT). The percentage of ZEA degradation ranged from 4% (24h) to 81% (48h). In the same conditions, α-ZOL and β-ZOL concentration decreased from 8% to 85%. No conversion of ZEA in α-ZOL and β-ZOL was detected. However, at 24h of exposure other degradation products of ZEA and its derived were detected. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Evaluation of CHO Benchmarks on the Arria 10 FPGA using Intel FPGA SDK for OpenCL

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Zheming [Argonne National Lab. (ANL), Argonne, IL (United States); Yoshii, Kazutomo [Argonne National Lab. (ANL), Argonne, IL (United States); Finkel, Hal [Argonne National Lab. (ANL), Argonne, IL (United States); Cappello, Franck [Argonne National Lab. (ANL), Argonne, IL (United States)

    2017-05-23

    The OpenCL standard is an open programming model for accelerating algorithms on heterogeneous computing system. OpenCL extends the C-based programming language for developing portable codes on different platforms such as CPU, Graphics processing units (GPUs), Digital Signal Processors (DSPs) and Field Programmable Gate Arrays (FPGAs). The Intel FPGA SDK for OpenCL is a suite of tools that allows developers to abstract away the complex FPGA-based development flow for a high-level software development flow. Users can focus on the design of hardware-accelerated kernel functions in OpenCL and then direct the tools to generate the low-level FPGA implementations. The approach makes the FPGA-based development more accessible to software users as the needs for hybrid computing using CPUs and FPGAs are increasing. It can also significantly reduce the hardware development time as users can evaluate different ideas with high-level language without deep FPGA domain knowledge. Benchmarking of OpenCL-based framework is an effective way for analyzing the performance of system by studying the execution of the benchmark applications. CHO is a suite of benchmark applications that provides support for OpenCL [1]. The authors presented CHO as an OpenCL port of the CHStone benchmark. Using Altera OpenCL (AOCL) compiler to synthesize the benchmark applications, they listed the resource usage and performance of each kernel that can be successfully synthesized by the compiler. In this report, we evaluate the resource usage and performance of the CHO benchmark applications using the Intel FPGA SDK for OpenCL and Nallatech 385A FPGA board that features an Arria 10 FPGA device. The focus of the report is to have a better understanding of the resource usage and performance of the kernel implementations using Arria-10 FPGA devices compared to Stratix-5 FPGA devices. In addition, we also gain knowledge about the limitations of the current compiler when it fails to synthesize a benchmark

  5. Vector-Tensor and Vector-Vector Decay Amplitude Analysis of B0->phi K*0

    CERN Document Server

    Aubert, B; Abrams, G S; Adye, T; Ahmed, S; Alam, M S; Albert, J; Aleksan, R; Allen, M T; Allison, J; Altenburg, D D; Andreotti, M; Angelini, C; Anulli, F; Arnaud, N; Asgeirsson, D J; Aston, D; Azzolini, V; Baak, M A; Back, J J; Baldini-Ferroli, R; Band, H R; Banerjee, Sw; Bard, D J; Barlow, N R; Barlow, R J; Barrett, M; Bartoldus, R; Batignani, G; Battaglia, M; Bauer, J M; Bechtle, P; Beck, T W; Behera, P K; Bellini, F; Benayoun, M; Benelli, G; Berger, N; Bernard, D; Berryhill, J W; Best, D S; Bettarini, S; Bettoni, D; Bevan, A J; Bhimji, W; Bhuyan, B; Bianchi, F; Biasini, M; Biesiada, J; Blanc, F; Blaylock, G; Blinov, V E; Bloom, P C; Blount, N L; Bomben, M; Bondioli, M; Bonneaud, G R; Bosisio, L; Boutigny, D; Bowerman, D A; Boyd, J T; Bozzi, C; Brandenburg, G; Brandt, T; Brau, J E; Briand, H; Brown, C M; Brown, D N; Bruinsma, M; Brunet, S; Bucci, F; Buchanan, C; Bugg, W; Bukin, A D; Bula, R; Burchat, P R; Burke, J P; Button-Shafer, J; Buzzo, A; Bóna, M; Cahn, R N; Calabrese, R; Calcaterra, A; Calderini, G; Campagnari, C; Carpinelli, M; Cartaro, C; Cavallo, N; Cavoto, G; Cenci, R; Chai, X; Chaisanguanthum, K S; Chao, M; Charles, E; Charles, M J; Chauveau, J; Chavez, C A; Chen, A; Chen, C; Chen, E; Chen, J C; Chen, S; Chen, X; Chen, X R; Cheng, B; Cheng, C H; Chia, Y M; Cibinetto, G; Clark, P J; Clarke, C K; Claus, R; Cochran, J; Coleman, J P; Contri, R; Convery, M R; Corwin, L A; Cossutti, F; Cottingham, W N; Couderc, F; Covarelli, R; Cowan, G; Cowan, R; Crawley, H B; Cremaldi, L; Cunha, A; Curry, S; Côté, D; D'Orazio, A; Dahmes, B; Dallapiccola, C; Danielson, N; Dasu, S; Datta, M; Dauncey, P D; David, P; Davier, M; Davis, C L; De Nardo, Gallieno; De Sangro, R; Del Amo-Sánchez, P; Del Buono, L; Del Re, D; Della Ricca, G; Denig, A G; Di Lodovico, F; Di Marco, E; Dingfelder, J C; Dittongo, S; Dong, L; Dorfan, J; Druzhinin, V P; Dubitzky, R S; Dubois-Felsmann, G P; Dujmic, D; Dunwoodie, W M; Dvoretskii, A; Ebert, M; Eckhart, E A; Eckmann, R; Edgar, C L; Edwards, A J; Egede, U; Eigen, G; Eisner, A M; Elmer, P; Emery, S; Ernst, J A; Eschenburg, V; Eschrich, I; Eyges, V; Fabozzi, F; Faccini, R; Fang, F; Feltresi, E; Ferrarotto, F; Ferroni, F; Field, R C; Finocchiaro, G; Flacco, C J; Flack, R L; Flächer, H U; Flood, K T; Ford, K E; Ford, W T; Forster, I J; Forti, F; Fortin, D; Foulkes, S D; Franek, B; Frey, R; Fritsch, M; Fry, J R; Fulsom, B G; Gabathuler, E; Gaidot, A; Gallo, F; Gamba, D; Gamet, R; Gan, K K; Ganzhur, S F; Gao, Y; Gary, J W; Gaspero, M; Gatto, C; Gaz, A; George, K A; Gill, M S; Giorgi, M A; Gladney, L; Glanzman, T; Godang, R; Golubev, V B; Gowdy, S J; Gradl, W; Graham, M T; Graugès-Pous, E; Grenier, P; Gritsan, A V; Grosdidier, G; Groysman, Y; Guo, Z J; Hadavand, H K; Haire, M; Halyo, V; Hamano, K; Hamel de Monchenault, G; Hamon, O; Harrison, P F; Harrison, T J; Hart, A J; Hartfiel, B L; Hast, C; Hauke, A; Hawkes, C M; Hearty, C; Held, T; Hertzbach, S S; Heusch, C A; Hill, E J; Hirschauer, J F; Hitlin, D G; Hollar, J J; Hong, T M; Honscheid, K; Hopkins, D A; Hrynóva, T; Hufnagel, D; Hulsbergen, W D; Hutchcroft, D E; Höcker, A; Igonkina, O; Innes, W R; Izen, J M; Jackson, P D; Jackson, P S; Jacobsen, R G; Jain, V; Jasper, H; Jawahery, A; Jessop, C P; Judd, D; Kadyk, J A; Kagan, H; Karyotakis, Yu; Kass, R; Kelsey, M H; Kerth, L T; Khan, A; Kim, H; Kim, P; Kirkby, D; Klose, V; Knecht, N S; Koch, H; Kolb, J A; Kolomensky, Yu G; Kovalskyi, D; Kowalewski, R V; Kozanecki, Witold; Kreisel, A; Krishnamurthy, M; Kroeger, R; Kroseberg, J; Kukartsev, G; Kutter, P E; Kyberd, P; La Vaissière, C de; Lacker, H M; Lae, C K; Lafferty, G D; Lanceri, L; Lange, D J; Lankford, A J; Latham, T E; Latour, E; Lau, Y P; Lazzaro, A; Le Diberder, F R; Lee, C L; Lees, J P; Legendre, M; Leith, D W G S; Lepeltier, V; Leruste, P; Lewandowski, B; Li Gioi, L; Li, S; Li, X; Lista, L; Liu, H; Lo Vetere, M; LoSecco, J M; Lockman, W S; Lombardo, V; Long, O; Lopes-Pegna, D; Lopez-March, N; Lou, X C; Lu, M; Luitz, S; Lund, P; Luppi, E; Lusiani, A; Lutz, A M; Lynch, G; Lynch, H L; Lü, C; Lüth, V; MacFarlane, D B; Macri, M M; Mader, W F; Majewski, S A; Malcles, J; Mallik, U; Mancinelli, G; Mandelkern, M A; Marchiori, G; Margoni, M; Marks, J; Marsiske, H; Martínez-Vidal, F; Mattison, T S; Mazur, M A; Mazzoni, M A; McKenna, J A; McMahon, T R; Mclachlin, S E; Meadows, B T; Mellado, B; Menges, W; Merkel, J; Messner, R; Meyer, N T; Meyer, W T; Mihályi, A; Mir, L M; Mishra, K; Mohanty, G B; Monge, M R; Monorchio, D; Moore, T B; Morandin, M; Morganti, M; Morganti, S; Morii, M; Muheim, F; Müller, D R; Nagel, M; Naisbit, M T; Narsky, I; Nash, J A; Nauenberg, U; Neal, H; Negrini, M; Neri, N; Nesom, G; Nicholson, H; Nikolich, M B; Nogowski, R; Nugent, I M; O'Grady, C P; Ocariz, J; Ofte, I; Olaiya, E O; Olivas, A; Olsen, J; Onuchin, A P; Orimoto, T J; Oyanguren, A; Ozcan, V E; Paar, H P; Pacetti, S; Palano, A; Palombo, F; Pan, B; Pan, Y; Panduro, W; Paoloni, E; Paolucci, P; Pappagallo, M; Park, W; Passaggio, S; Patel, P M; Patrignani, C; Patteri, P; Payne, D J; Pelizaeus, M; Perazzo, A; Perl, M; Peruzzi, I M; Peters, K; Petersen, B A; Petrella, A; Petzold, A; Piatenko, T; Piccolo, D; Piccolo, M; Piemontese, L; Pierini, M; Piredda, G; Playfer, S; Poireau, V; Polci, F; Pompili, A; Porter, F C; Posocco, M; Potter, C T; Prell, S; Prencipe, E; Prepost, R; Pripstein, M; Pruvot, S; Pulliam, T; Purohit, M V; Qi, N D; Rahatlou, S; Rahimi, A M; Rahmat, R; Rama, M; Ratcliff, B N; Raven, G; Regensburger, J J; Ricciardi, S; Richman, J D; Ritchie, J L; Rizzo, G; Roberts, D A; Robertson, A I; Robertson, S H; Robutti, E; Rodier, S; Roe, N A; Ronan, M T; Roney, J M; Rong, G; Roodman, A; Roos, L; Rosenberg, E I; Rotondo, M; Roudeau, P; Rubin, A E; Ruddick, W O; Röthel, W; Sacco, R; Saeed, M A; Safai-Tehrani, F; Saleem, M; Salnikov, A A; Salvatore, F; Sanders, D A; Santroni, A; Saremi, S; Satpathy, A; Schalk, T; Schenk, S; Schilling, C J; Schindler, R H; Schofield, K C; Schott, G; Schröder, T; Schröder, H; Schubert, J; Schubert, K R; Schumm, B A; Schune, M H; Schwiening, J; Schwierz, R; Schwitters, R F; Sciacca, C; Sciolla, G; Seiden, A; Sekula, S J; Serednyakov, S I; Serrano, J; Sharma, V; Shen, B C; Sherwood, D J; Simard, M; Simi, G; Simonetto, F; Sinev, N B; Skovpen, Yu I; Smith, A J S; Smith, J G; Snoek, H L; Snyder, A; Sobie, R J; Soffer, A; Sokoloff, M D; Solodov, E P; Spaan, B; Spanier, S M; Spitznagel, M; Spradlin, P; Steinke, M; Stelzer, J; Stocchi, A; Stoker, D P; Stroili, R; Strom, D; Strube, J; Stugu, B; Stängle, H; Su, D; Sullivan, M K; Summers, D J; Sundermann, J E; Suzuki, K; Swain, S K; Taras, P; Taylor, F; Telnov, A V; Teodorescu, L; Ter-Antonian, R; Therin, G; Thiebaux, C; Thompson, J M; Tisserand, V; Todyshev, Y K; Toki, W H; Torrence, E; Tosi, S; Touramanis, C; Ulmer, K A; Uwer, U; Van Bakel, N; Vasseur, G; Vavra, J; Vazquez, A; Verderi, M; Viaud, F B; Vitale, L; Voci, C; Voena, C; Volk, A; Wagner, A P; Wagner, S R; Wagoner, D E; Waldi, R; Walker, D; Walsh, J J; Wang, K; Wang, P; Wang, W F; Wappler, F R; Watson, A T; Weaver, M; Weinstein, A J R; Wenzel, W A; Wilden, L; Williams, D C; Williams, J C; Wilson, F F; Wilson, J R; Wilson, M G; Wilson, R J; Winklmeier, F; Wisniewski, W J; Wittgen, M; Wong, Q K; Wormser, G; Wren, A C; Wright, D H; Wright, D M; Wu, J; Wu, S L; Wulsin, H W; Xie, Y; Yamamoto, R K; Yarritu, A K; Ye, S; Yi, J I; Yi, K; Young, C C; Yu, Z; Yéche, C; Zain, S B; Zallo, A; Zeng, Q; Zghiche, A; Zhang, J; Zhang, L; Zhao, H W; Zhu, Y S; Ziegler, V; Zito, M; Çuhadar-Dönszelmann, T; al, et

    2007-01-01

    We perform an amplitude analysis of the decays B0->phi K^*_2(1430)0, phi K^*(892)0, and phi(K pi)^0_S-wave with a sample of about 384 million BBbar pairs recorded with the BABAR detector. The fractions of longitudinal polarization f_L of the vector-tensor and vector-vector decay modes are measured to be 0.853 +0.061-0.069 +-0.036 and 0.506 +-0.040 +-0.015, respectively. Overall, twelve parameters are measured for the vector-vector decay and seven parameters for the vector-tensor decay, including the branching fractions and parameters sensitive to CP-violation.

  6. Label-free live cell imaging by Confocal Raman Microscopy identifies CHO host and producer cell lines.

    Science.gov (United States)

    Prats Mateu, Batirtze; Harreither, Eva; Schosserer, Markus; Puxbaum, Verena; Gludovacz, Elisabeth; Borth, Nicole; Gierlinger, Notburga; Grillari, Johannes

    2017-01-01

    As a possible viable and non-invasive method to identify high producing cells, Confocal Raman Microscopy was shown to be able to differentiate CHO host cell lines and derivative production clones. Cluster analysis of spectra and their derivatives was able to differentiate between different producer cell lines and a host, and also distinguished between an intracellular region of high lipid and protein content that in structure resembles the Endoplasmic Reticulum. This ability to identify the ER may be a major contributor to the identification of high producers. PCA enabled the discrimination even of host cell lines and their subclones with inherently higher production capacity. The method is thus a promising option that may contribute to early, non-invasive identification of high potential candidates during cell line development and possibly could also be used for proof of identity of established production clones. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Transcriptional Silencing of Retroviral Vectors

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M.; Pedersen, F.S.

    1996-01-01

    Although retroviral vector systems have been found to efficiently transduce a variety of cell types in vitro, the use of vectors based on murine leukemia virus in preclinical models of somatic gene therapy has led to the identification of transcriptional silencing in vivo as an important problem...

  8. On the Witt vector Frobenius

    DEFF Research Database (Denmark)

    Davis, Christopher James; Kedlaya, Kiran

    2014-01-01

    We study the kernel and cokernel of the Frobenius map on the p-typical Witt vectors of a commutative ring, not necessarily of characteristic p. We give many equivalent conditions to surjectivity of the Frobenius map on both finite and infinite length Witt vectors. In particular, surjectivity on f...

  9. Estimation of Motion Vector Fields

    DEFF Research Database (Denmark)

    Larsen, Rasmus

    1993-01-01

    This paper presents an approach to the estimation of 2-D motion vector fields from time varying image sequences. We use a piecewise smooth model based on coupled vector/binary Markov random fields. We find the maximum a posteriori solution by simulated annealing. The algorithm generate sample...

  10. Vectors on the Basketball Court

    Science.gov (United States)

    Bergman, Daniel

    2010-01-01

    An Idea Bank published in the April/May 2009 issue of "The Science Teacher" describes an experiential physics lesson on vectors and vector addition (Brown 2009). Like its football predecessor, the basketball-based investigation presented in this Idea Bank addresses National Science Education Standards Content B, Physical Science, 9-12 (NRC 1996)…

  11. The Neural Support Vector Machine

    NARCIS (Netherlands)

    Wiering, Marco; van der Ree, Michiel; Embrechts, Mark; Stollenga, Marijn; Meijster, Arnold; Nolte, A; Schomaker, Lambertus

    2013-01-01

    This paper describes a new machine learning algorithm for regression and dimensionality reduction tasks. The Neural Support Vector Machine (NSVM) is a hybrid learning algorithm consisting of neural networks and support vector machines (SVMs). The output of the NSVM is given by SVMs that take a

  12. GPU Accelerated Vector Median Filter

    Science.gov (United States)

    Aras, Rifat; Shen, Yuzhong

    2011-01-01

    Noise reduction is an important step for most image processing tasks. For three channel color images, a widely used technique is vector median filter in which color values of pixels are treated as 3-component vectors. Vector median filters are computationally expensive; for a window size of n x n, each of the n(sup 2) vectors has to be compared with other n(sup 2) - 1 vectors in distances. General purpose computation on graphics processing units (GPUs) is the paradigm of utilizing high-performance many-core GPU architectures for computation tasks that are normally handled by CPUs. In this work. NVIDIA's Compute Unified Device Architecture (CUDA) paradigm is used to accelerate vector median filtering. which has to the best of our knowledge never been done before. The performance of GPU accelerated vector median filter is compared to that of the CPU and MPI-based versions for different image and window sizes, Initial findings of the study showed 100x improvement of performance of vector median filter implementation on GPUs over CPU implementations and further speed-up is expected after more extensive optimizations of the GPU algorithm .

  13. Developement of serum-free media in CHO-DG44 cells using a central composite statistical design.

    Science.gov (United States)

    Parampalli, Ananth; Eskridge, Kent; Smith, Leonard; Meagher, Michael M; Mowry, Mark C; Subramanian, Anuradha

    2007-05-01

    A serum free medium was developed for the production of recombinant antibody against Botulinum A (BoNTA) using dihydrofolate reductase deficient Chinese Hamster Ovary Cells (CHO-DG44) in suspension culture. An initial control basal medium was prepared, which was similar in composition to HAM's F12: IMDM (1:1) supplemented with insulin, transeferrin, selenium and a lipid mixture. The vitamin concentration of the basal medium was twice that of HAM's F12: IMDM (1:1). CHO-DG44 cells expressing S25 antibody grew from 2 x 10(5) cells to maximum cell density of 1.04 x 10(6) cells/ml after 5 days in this control medium. A central composite design was used to identify optimal levels and interaction among five groups of medium components. These five groups were glutamine, Essential Amino Acids (EAA), Non Essential Amino Acids (NEAA), Insulin, Transferrin, Selenium (ITS), and lipids. Fifty experiments were carried out in four batches, with two controls in each batch. There was little effect of ITS and Lipid concentrations over the range studied, and glutamine concentration showed a strong interaction with EAA. The optimal concentrations of the variables studied were 2.5 mM Glutamine, 7.4 mM (2x) EAA, 1.4 mM (0.5x) NEAA, 1x ITS supplement, 0.7x Lipids supplement. The maximum viable cell density attained in the optimized medium was 1.4 x 10(6) cells/ml, a 35% improvement over the control culture, while the final antibody titer attained was 22 +/- 3.4 mug/mL, a 50% improvement.

  14. Effect of Lippia alba and Cymbopogon citratus essential oils on biofilms of Streptococcus mutans and cytotoxicity in CHO cells.

    Science.gov (United States)

    Tofiño-Rivera, A; Ortega-Cuadros, M; Galvis-Pareja, D; Jiménez-Rios, H; Merini, L J; Martínez-Pabón, M C

    2016-12-24

    Caries is a public health problem, given that it prevails in 60 to 90% of the school-age global population. Multiple factors interact in its etiology, among them dental plaque is necessary to have lactic acid producing microorganisms like Streptococcus from he Mutans group. Existing prevention and treatment measures are not totally effective and generate adverse effects, which is why it is necessary to search for complementary strategies for their management. The study sought to evaluate the eradication capacity of Streptococcus mutans biofilms and the toxicity on eukaryotic cells of Lippia alba and Cymbopogon citratus essential oils. Essential oils were extracted from plant material through steam distillation and then its chemical composition was determined. The MBEC-high-throughput (MBEC-HTP) (Innovotech, Edmonton, Alberta, Canada) assay used to determine the eradication concentration of S. mutans ATCC 35668 strain biofilms. Cytotoxicity was evaluated on CHO cells through the MTT cell proliferation assay. The major components in both oils were Geraniol and Citral; in L. alba 18.9% and 15.9%, respectively, and in C. citratus 31.3% and 26.7%. The L. alba essential oils presented eradication activity against S. mutans biofilms of 95.8% in 0.01mg/dL concentration and C. citratus essential oils showed said eradication activity of 95.4% at 0.1, 0.01mg/dL concentrations and of 93.1% in the 0.001mg/dL concentration; none of the concentrations of both essential oils showed toxicity on CHO cells during 24h. The L. alba and C. citratus essential oils showed eradication activity against S. mutans biofilms and null cytotoxicity, evidencing the need to conduct further studies that can identify their active components and in order to guide a safe use in treating and preventing dental caries. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  15. Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

    LENUS (Irish Health Repository)

    Burleigh, Susan C

    2011-10-18

    Abstract Background The glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins. Results Chinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG. Conclusions The use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry.

  16. Photochemical formation of HCO and CH3 on the ground S0 (1A') state of CH3CHO.

    Science.gov (United States)

    Heazlewood, Brianna R; Rowling, Steven J; Maccarone, Alan T; Jordan, Meredith J T; Kable, Scott H

    2009-02-07

    The dynamics of the photodissociation of CH(3)CHO into CH(3) + HCO products have been investigated at energies between 30,953 and 31,771 cm(-1), spanning the threshold for radical production on the triplet (T(1)) surface. A barrierless pathway to CH(3) + HCO radical products formed on the ground state (S(0)) surface was discovered and established to be an important reaction channel in acetaldehyde photodissociation throughout this wavelength range. HCO laser induced fluorescence (LIF) spectra recorded from CH(3)CHO dissociated above and below the T(1) barrier energy are quite different; HCO produced on S(0) yields a more congested LIF spectrum with sharp rotational transitions, while HCO formed on the T(1) surface displays fewer, more intense, Doppler-broadened lines. These differences have been further explored in the populations of the HCO K(a) = 1 doublets. Despite the upper and lower levels being almost isoenergetic, HCO formed on T(1) preferentially populates the upper K(c) state due to the geometry of the T(1) transition state structure. In contrast, HCO formed on S(0) produces equal population in each of the upper and lower K(a) = 1 components. Product state distributions (PSDs) showed that HCO formed on S(0) is born with an approximately statistical distribution of population in the available product states, modeled well by phase space theory. HCO formed on the T(1) surface, in contrast, has a PSD that can be characterized as arising from "impulsive" dynamics. Previous discrepancies in the height of the T(1) barrier are discussed following the observation that, once the T(1) channel is energetically accessible, there is competition between the S(0) and T(1) pathways, with the dominance of the triplet channel increasing with increasing photolysis energy.

  17. Reprogramming amino acid catabolism in CHO cells with CRISPR-Cas9 genome editing improves cell growth and reduces by-product secretion

    DEFF Research Database (Denmark)

    Ley, Daniel; Pereira, Sara; Pedersen, Lasse Ebdrup

    2017-01-01

    CHO cells primarily utilize amino acids for three processes: biomass synthesis, recombinant protein production and catabolism. In this work, we disrupted 9 amino acid catabolic genes participating in 7 dierent catabolic pathways, to increase synthesis of biomass and recombinant protein, while red...

  18. Development and validation of a high-content screening in vitro micronucleus assay in CHO-k1 and HepG2 cells

    NARCIS (Netherlands)

    Westerink, W.M.; Schirris, T.J.J.; Horbach, G.J.; Schoonen, W.G.

    2011-01-01

    In the present study an automated image analysis assisted in vitro micronucleus assay was developed with the rodent cell line CHO-k1 and the human hepatoma cell line HepG2, which are both commonly used in regulatory genotoxicity assays. The HepG2 cell line was chosen because of the presence in these

  19. Antigenotoxicity of Agaricus blazei mushroom organic and aqueous extracts in chromosomal aberration and cytokinesis block micronucleus assays in CHO-k1 and HTC cells.

    Science.gov (United States)

    Bellini, M F; Angeli, J P F; Matuo, R; Terezan, A P; Ribeiro, L R; Mantovani, M S

    2006-04-01

    Agaricus blazei (Ab) has become popularly known for its medicinal properties. Scientifically, it has been tested with regard to its capacity to protect genetic material against damage. We examined different organic extracts (methanolic extract -- ME, hexanic extract -- HE and n-butanolic extract -- BE) and an aqueous extract (AE) of Ab, for their capacity to induce DNA damage as well as for their protective effect. Genetic damage was determined by the chromosomal aberration assay (CA) in CHO-k1 cells for all extracts and the cytokinesis block micronucleus assay (CBMN) in non drug-metabolizing (CHO-k1) and drug-metabolizing (HTC) cell lines for extract BE only. The extracts did not show clastogenicity but showed anticlastogenicity. The greatest percent reduction obtained were with BE (105%) and AE (126%) treatments in CA. BE treatment did not display genotoxicity in CHO-k1, but was genotoxic in HTC. However, BE was shown to be antigenotoxic causing decreased micronucleus frequency in HTC and CHO-k1 cells. These results suggest that all the extracts contained protective substances, but in some cases they could show a genotoxic effect with regard to metabolism. Therefore, these findings warrant caution in the use of this mushroom by the population.

  20. Emerging vector borne diseases – incidence through vectors

    Directory of Open Access Journals (Sweden)

    Sara eSavic

    2014-12-01

    Full Text Available Vector borne diseases use to be a major public health concern only in tropical and subtropical areas, but today they are an emerging threat for the continental and developed countries also. Nowdays, in intercontinetal countries, there is a struggle with emerging diseases which have found their way to appear through vectors. Vector borne zoonotic diseases occur when vectors, animal hosts, climate conditions, pathogens and susceptible human population exist at the same time, at the same place. Global climate change is predicted to lead to an increase in vector borne infectious diseases and disease outbreaks. It could affect the range and popultion of pathogens, host and vectors, transmission season, etc. Reliable surveilance for diseases that are most likely to emerge is required. Canine vector borne diseases represent a complex group of diseases including anaplasmosis, babesiosis, bartonellosis, borreliosis, dirofilariosis, erlichiosis, leishmaniosis. Some of these diseases cause serious clinical symptoms in dogs and some of them have a zoonotic potential with an effect to public health. It is expected from veterinarians in coordination with medical doctors to play a fudamental role at primeraly prevention and then treatment of vector borne diseases in dogs. The One Health concept has to be integrated into the struggle against emerging diseases.During a four year period, from 2009-2013, a total number of 551 dog samples were analysed for vector borne diseases (borreliosis, babesiosis, erlichiosis, anaplasmosis, dirofilariosis and leishmaniasis in routine laboratory work. The analysis were done by serological tests – ELISA for borreliosis, dirofilariosis and leishmaniasis, modified Knott test for dirofilariosis and blood smear for babesiosis, erlichiosis and anaplasmosis. This number of samples represented 75% of total number of samples that were sent for analysis for different diseases in dogs. Annually, on avarege more then half of the samples

  1. Hydrolysis of Phosphatidylcholine-Isoprostanes (PtdCho-IP) by Peripheral Human Group IIA, V and X Secretory Phospholipases A2 (sPLA2).

    Science.gov (United States)

    Kuksis, Arnis; Pruzanski, Waldemar

    2017-06-01

    Biologically active F- and E/D-type-prostane ring isomers (F2-IP and E2/D2-IP, respectively) are produced in situ by non-enzymatic peroxidation of arachidonic acid esterified to GroPCho (PtdCho-IP) and are universally distributed in tissue lipoproteins and cell membranes. Previous work has shown that platelet-activating factor acetylhydrolases (PAF-AH) are the main endogenous PLA2 involved in degradation of PtdCho-IP. The present study shows that the PtdCho-IP are also subject to hydrolysis by group IIA, V and X secretory PLA2, which also have a wide peripheral tissue distribution. For this demonstration, we compared the LC/MS profiles of PtdCho-IP of auto-oxidized plasma lipoproteins after incubation for 1-4 h (37 °C) in the absence or presence of recombinant human sPLA2 (1-2.5 µg/ml). In the absence of exogenously added sPLA2 the total PtdCho-IP level after 4 h incubation reached 15.9, 21.6 and 8.7 nmol/mg protein of LDL, HDL and HDL3, respectively. In the presence of group V or group X sPLA2 (2.5 µg/ml), the PtdCho-IP was completely hydrolyzed in 1 h, while in the presence of group IIA sPLA2 (2.5 µg/ml) the hydrolysis was less than 25% in 4 h, although it was complete after 8-24 h incubation. This report provides the first demonstration that PtdCho-IP are readily hydrolyzed by group IIA, V and X sPLA2. A co-location of sPLA2 and the substrates in various tissues has been recorded. Thus, the initiation of interaction and production of isoprostanes in situ are highly probable.

  2. Validated detection of human anti-chimeric immune responses in serum of neuroblastoma patients treated with ch14.18/CHO.

    Science.gov (United States)

    Siebert, Nikolai; Eger, Christin; Seidel, Diana; Jüttner, Madlen; Lode, Holger N

    2014-05-01

    Human/mouse chimeric monoclonal antibody (mAb) ch14.18/CHO is directed against disialoganglioside GD2. Activity and efficacy of this mAb are currently determined in ongoing clinical Phase II and -III studies in high-risk neuroblastoma (NB). Based on the chimeric nature of this mAb, some patients may develop a human anti-chimeric immune response (Mirick et al., 2004) which impacts on pharmacokinetics and may induce anti-anti-idiotype (Id) mAb with a potential survival benefit. Therefore, a validated method of quantitative detection of human anti-chimeric antibodies (HACA) in serum samples of NB patients treated with ch14.18/CHO is an important tool for monitoring of clinical trials. Here, we report a validated sandwich enzyme-linked immunosorbent assay (ELISA) according to the one arm binding principle using ch14.18/CHO as a capture mAb and biotinylated ch14.18/CHO mAb for detection. Ganglidiomab, a monoclonal anti-Id Ab to ch14.18/CHO (Lode et al., 2013), was used as a standard for assay validation and HACA quantification. Systematic evaluation of the established ELISA procedure revealed an optimal serum sample dilution factor of 1:160. Assay validation was accomplished with a set of tailored quality controls (QC) containing distinct concentrations of ganglidiomab (3 and 15μg/ml). The coefficients of variation (CV) for all within-assay and inter-assay measurements using QCs were under 20% and the limit of detection (LOD) was 1.1μg/ml. Three patients (P1, P2, P3) treated with a 10day continuous infusion of 100mg/m(2) of ch14.18/CHO were selected for analysis with this assay. Selection was based on ch14.18/CHO drug level on day 8 in cycle 2 of >10μg/ml (expected) (P1) and of <2μg/ml (unexpected) (P2 and P3). Both patients with unexpected low ch14.18/CHO levels revealed a strong signal in the HACA ELISA. Interestingly, ch14.18/CHO-mediated complement-dependent cytotoxicity (CDC) could not be detected in P2 in contrast to P3 suggesting anti-NB activity even in the

  3. Stable piecewise polynomial vector fields

    Directory of Open Access Journals (Sweden)

    Claudio Pessoa

    2012-09-01

    Full Text Available Let $N={y>0}$ and $S={y<0}$ be the semi-planes of $mathbb{R}^2$ having as common boundary the line $D={y=0}$. Let $X$ and $Y$ be polynomial vector fields defined in $N$ and $S$, respectively, leading to a discontinuous piecewise polynomial vector field $Z=(X,Y$. This work pursues the stability and the transition analysis of solutions of $Z$ between $N$ and $S$, started by Filippov (1988 and Kozlova (1984 and reformulated by Sotomayor-Teixeira (1995 in terms of the regularization method. This method consists in analyzing a one parameter family of continuous vector fields $Z_{epsilon}$, defined by averaging $X$ and $Y$. This family approaches $Z$ when the parameter goes to zero. The results of Sotomayor-Teixeira and Sotomayor-Machado (2002 providing conditions on $(X,Y$ for the regularized vector fields to be structurally stable on planar compact connected regions are extended to discontinuous piecewise polynomial vector fields on $mathbb{R}^2$. Pertinent genericity results for vector fields satisfying the above stability conditions are also extended to the present case. A procedure for the study of discontinuous piecewise vector fields at infinity through a compactification is proposed here.

  4. Chikungunya Virus–Vector Interactions

    Directory of Open Access Journals (Sweden)

    Lark L. Coffey

    2014-11-01

    Full Text Available Chikungunya virus (CHIKV is a mosquito-borne alphavirus that causes chikungunya fever, a severe, debilitating disease that often produces chronic arthralgia. Since 2004, CHIKV has emerged in Africa, Indian Ocean islands, Asia, Europe, and the Americas, causing millions of human infections. Central to understanding CHIKV emergence is knowledge of the natural ecology of transmission and vector infection dynamics. This review presents current understanding of CHIKV infection dynamics in mosquito vectors and its relationship to human disease emergence. The following topics are reviewed: CHIKV infection and vector life history traits including transmission cycles, genetic origins, distribution, emergence and spread, dispersal, vector competence, vector immunity and microbial interactions, and co-infection by CHIKV and other arboviruses. The genetics of vector susceptibility and host range changes, population heterogeneity and selection for the fittest viral genomes, dual host cycling and its impact on CHIKV adaptation, viral bottlenecks and intrahost diversity, and adaptive constraints on CHIKV evolution are also discussed. The potential for CHIKV re-emergence and expansion into new areas and prospects for prevention via vector control are also briefly reviewed.

  5. Chikungunya Virus–Vector Interactions

    Science.gov (United States)

    Coffey, Lark L.; Failloux, Anna-Bella; Weaver, Scott C.

    2014-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes chikungunya fever, a severe, debilitating disease that often produces chronic arthralgia. Since 2004, CHIKV has emerged in Africa, Indian Ocean islands, Asia, Europe, and the Americas, causing millions of human infections. Central to understanding CHIKV emergence is knowledge of the natural ecology of transmission and vector infection dynamics. This review presents current understanding of CHIKV infection dynamics in mosquito vectors and its relationship to human disease emergence. The following topics are reviewed: CHIKV infection and vector life history traits including transmission cycles, genetic origins, distribution, emergence and spread, dispersal, vector competence, vector immunity and microbial interactions, and co-infection by CHIKV and other arboviruses. The genetics of vector susceptibility and host range changes, population heterogeneity and selection for the fittest viral genomes, dual host cycling and its impact on CHIKV adaptation, viral bottlenecks and intrahost diversity, and adaptive constraints on CHIKV evolution are also discussed. The potential for CHIKV re-emergence and expansion into new areas and prospects for prevention via vector control are also briefly reviewed. PMID:25421891

  6. Emerging Vector-Borne Diseases - Incidence through Vectors.

    Science.gov (United States)

    Savić, Sara; Vidić, Branka; Grgić, Zivoslav; Potkonjak, Aleksandar; Spasojevic, Ljubica

    2014-01-01

    Vector-borne diseases use to be a major public health concern only in tropical and subtropical areas, but today they are an emerging threat for the continental and developed countries also. Nowadays, in intercontinental countries, there is a struggle with emerging diseases, which have found their way to appear through vectors. Vector-borne zoonotic diseases occur when vectors, animal hosts, climate conditions, pathogens, and susceptible human population exist at the same time, at the same place. Global climate change is predicted to lead to an increase in vector-borne infectious diseases and disease outbreaks. It could affect the range and population of pathogens, host and vectors, transmission season, etc. Reliable surveillance for diseases that are most likely to emerge is required. Canine vector-borne diseases represent a complex group of diseases including anaplasmosis, babesiosis, bartonellosis, borreliosis, dirofilariosis, ehrlichiosis, and leishmaniosis. Some of these diseases cause serious clinical symptoms in dogs and some of them have a zoonotic potential with an effect to public health. It is expected from veterinarians in coordination with medical doctors to play a fundamental role at primarily prevention and then treatment of vector-borne diseases in dogs. The One Health concept has to be integrated into the struggle against emerging diseases. During a 4-year period, from 2009 to 2013, a total number of 551 dog samples were analyzed for vector-borne diseases (borreliosis, babesiosis, ehrlichiosis, anaplasmosis, dirofilariosis, and leishmaniasis) in routine laboratory work. The analysis was done by serological tests - ELISA for borreliosis, dirofilariosis, and leishmaniasis, modified Knott test for dirofilariosis, and blood smear for babesiosis, ehrlichiosis, and anaplasmosis. This number of samples represented 75% of total number of samples that were sent for analysis for different diseases in dogs. Annually, on average more then half of the samples

  7. Vector control of induction machines

    CERN Document Server

    Robyns, Benoit

    2012-01-01

    After a brief introduction to the main law of physics and fundamental concepts inherent in electromechanical conversion, ""Vector Control of Induction Machines"" introduces the standard mathematical models for induction machines - whichever rotor technology is used - as well as several squirrel-cage induction machine vector-control strategies. The use of causal ordering graphs allows systematization of the design stage, as well as standardization of the structure of control devices. ""Vector Control of Induction Machines"" suggests a unique approach aimed at reducing parameter sensitivity for

  8. Vector boson scattering at CLIC

    Energy Technology Data Exchange (ETDEWEB)

    Kilian, Wolfgang; Fleper, Christian [Department Physik, Universitaet Siegen, 57068 Siegen (Germany); Reuter, Juergen [DESY Theory Group, 22603 Hamburg (Germany); Sekulla, Marco [Institut fuer Theoretische Physik, Karlsruher Institut fuer Technologie, 76131 Karlsruhe (Germany)

    2016-07-01

    Linear colliders operating in a range of multiple TeV are able to investigate the details of vector boson scattering and electroweak symmetry breaking. We calculate cross sections with the Monte Carlo generator WHIZARD for vector boson scattering processes at the future linear e{sup +} e{sup -} collider CLIC. By finding suitable cuts, the vector boson scattering signal processes are isolated from the background. Finally, we are able to determine exclusion sensitivities on the non-Standard Model parameters of the relevant dimension eight operators.

  9. Vector independent transmission of the vector-borne bluetongue virus.

    Science.gov (United States)

    van der Sluijs, Mirjam Tineke Willemijn; de Smit, Abraham J; Moormann, Rob J M

    2016-01-01

    Bluetongue is an economically important disease of ruminants. The causative agent, Bluetongue virus (BTV), is mainly transmitted by insect vectors. This review focuses on vector-free BTV transmission, and its epizootic and economic consequences. Vector-free transmission can either be vertical, from dam to fetus, or horizontal via direct contract. For several BTV-serotypes, vertical (transplacental) transmission has been described, resulting in severe congenital malformations. Transplacental transmission had been mainly associated with live vaccine strains. Yet, the European BTV-8 strain demonstrated a high incidence of transplacental transmission in natural circumstances. The relevance of transplacental transmission for the epizootiology is considered limited, especially in enzootic areas. However, transplacental transmission can have a substantial economic impact due to the loss of progeny. Inactivated vaccines have demonstrated to prevent transplacental transmission. Vector-free horizontal transmission has also been demonstrated. Since direct horizontal transmission requires close contact of animals, it is considered only relevant for within-farm spreading of BTV. The genetic determinants which enable vector-free transmission are present in virus strains circulating in the field. More research into the genetic changes which enable vector-free transmission is essential to better evaluate the risks associated with outbreaks of new BTV serotypes and to design more appropriate control measures.

  10. Introduction to matrices and vectors

    CERN Document Server

    Schwartz, Jacob T

    2001-01-01

    In this concise undergraduate text, the first three chapters present the basics of matrices - in later chapters the author shows how to use vectors and matrices to solve systems of linear equations. 1961 edition.

  11. All optical vector magnetometer Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This Phase I research project will investigate a novel method of operating an atomic magnetometer to simultaneously measure total magnetic fields and vector magnetic...

  12. GRE Enzymes for Vector Analysis

    Data.gov (United States)

    U.S. Environmental Protection Agency — Microbial enzyme data that were collected during the 2004-2006 EMAP-GRE program. These data were then used by Moorhead et al (2016) in their ecoenzyme vector...

  13. A Note on Vector Bimeasures.

    Science.gov (United States)

    1987-11-01

    Phrases: N/A ] 19 RCT lontilue on revrm if necozsary and identify by block number) •"SA Fubini type theorem is obtained for vector bimasure integrals...Abstract A Fubini type theorem is obtained for vector bimeasure integrals. AMS (1980) subject classification: Primary 28B05; Secondary 60G12...Ylinen [11]. In the works mentioned above the authors consistently impose, in their definition of integrability. a Fubini type condition which cannot

  14. Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor.

    Science.gov (United States)

    Zhang, Ye; Stobbe, Per; Silvander, Christian Orrego; Chotteau, Véronique

    2015-11-10

    Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200 pF/cm (where 1 pF/cm is equivalent to 1 × 10(6)viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37 °C to 29 °C was studied during 13 days at cell densities above 100 pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31 °C was applied at cell density 100 pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period. Copyright © 2015 The Authors. Published by Elsevier B.V. All

  15. Integrated cell and process engineering for improved transient production of a "difficult-to-express" fusion protein by CHO cells.

    Science.gov (United States)

    Johari, Yusuf B; Estes, Scott D; Alves, Christina S; Sinacore, Marty S; James, David C

    2015-12-01

    Based on an optimized electroporation protocol, we designed a rapid, milliliter-scale diagnostic transient production assay to identify limitations in the ability of Chinese hamster ovary (CHO) cells to produce a model "difficult-to-express" homodimeric Fc-fusion protein, Sp35Fc, that exhibited very low volumetric titer and intracellular formation of disulfide-bonded oligomeric aggregates post-transfection. As expression of Sp35Fc induced an unfolded protein response in transfected host cells, we utilized the transient assay to compare, in parallel, multiple functionally diverse strategies to engineer intracellular processing of Sp35Fc in order to increase production and reduce aggregation as two discrete design objectives. Specifically, we compared the effect of (i) co-expression of ER-resident molecular chaperones (BiP, PDI, CypB) or active forms of UPR transactivators (ATF6c, XBP1s) at varying recombinant gene load, (ii) addition of small molecules known to act as chemical chaperones (PBA, DMSO, glycerol, betaine, TMAO) or modulate UPR signaling (PERK inhibitor GSK2606414) at varying concentration, (iii) a reduction in culture temperature to 32°C. Using this information, we designed a biphasic, Sp35Fc-specific transient manufacturing process mediated by lipofection that utilized CypB co-expression at an optimal Sp35Fc:CypB gene ratio of 5:1 to initially maximize transfected cell proliferation, followed by addition of a combination of PBA (0.5 mM) and glycerol (1% v/v) at the onset of stationary phase to maximize cell specific production and eliminate Sp35Fc aggregation. Using this optimal, engineered process transient Sp35Fc production was significantly increased sixfold over a 12 day production process with no evidence of disulfide-bonded aggregates. Finally, transient production in clonally derived sub-populations (derived from parental CHO host) screened for a heritably improved capability to produce Sp35Fc was also significantly improved by the optimized

  16. VectorBase: a home for invertebrate vectors of human pathogens

    Science.gov (United States)

    Lawson, Daniel; Arensburger, Peter; Atkinson, Peter; Besansky, Nora J.; Bruggner, Robert V.; Butler, Ryan; Campbell, Kathryn S.; Christophides, George K.; Christley, Scott; Dialynas, Emmanuel; Emmert, David; Hammond, Martin; Hill, Catherine A.; Kennedy, Ryan C.; Lobo, Neil F.; MacCallum, M. Robert; Madey, Greg; Megy, Karine; Redmond, Seth; Russo, Susan; Severson, David W.; Stinson, Eric O.; Topalis, Pantelis; Zdobnov, Evgeny M.; Birney, Ewan; Gelbart, William M.; Kafatos, Fotis C.; Louis, Christos; Collins, Frank H.

    2007-01-01

    VectorBase () is a web-accessible data repository for information about invertebrate vectors of human pathogens. VectorBase annotates and maintains vector genomes providing an integrated resource for the research community. Currently, VectorBase contains genome information for two organisms: Anopheles gambiae, a vector for the Plasmodium protozoan agent causing malaria, and Aedes aegypti, a vector for the flaviviral agents causing Yellow fever and Dengue fever. PMID:17145709

  17. Decays of the vector glueball

    Science.gov (United States)

    Giacosa, Francesco; Sammet, Julia; Janowski, Stanislaus

    2017-06-01

    We calculate two- and three-body decays of the (lightest) vector glueball into (pseudo)scalar, (axial-)vector, as well as pseudovector and excited vector mesons in the framework of a model of QCD. While absolute values of widths cannot be predicted because the corresponding coupling constants are unknown, some interesting branching ratios can be evaluated by setting the mass of the yet hypothetical vector glueball to 3.8 GeV as predicted by quenched lattice QCD. We find that the decay mode ω π π should be one of the largest (both through the decay chain O →b1π →ω π π and through the direct coupling O →ω π π ). Similarly, the (direct and indirect) decay into π K K*(892 ) is sizable. Moreover, the decays into ρ π and K*(892 )K are, although subleading, possible and could play a role in explaining the ρ π puzzle of the charmonium state ψ (2 S ) thanks to a (small) mixing with the vector glueball. The vector glueball can be directly formed at the ongoing BESIII experiment as well as at the future PANDA experiment at the FAIR facility. If the width is sufficiently small (≲100 MeV ) it should not escape future detection. It should be stressed that the employed model is based on some inputs and simplifying assumptions: the value of glueball mass (at present, the quenched lattice value is used), the lack of mixing of the glueball with other quarkonium states, and the use of few interaction terms. It then represents a first step toward the identification of the main decay channels of the vector glueball, but shall be improved when corresponding experimental candidates and/or new lattice results will be available.

  18. The biological control of disease vectors.

    Science.gov (United States)

    Okamoto, Kenichi W; Amarasekare, Priyanga

    2012-09-21

    Vector-borne diseases are common in nature and can have a large impact on humans, livestock and crops. Biological control of vectors using natural enemies or competitors can reduce vector density and hence disease transmission. However, the indirect interactions inherent in host-vector disease systems make it difficult to use traditional pest control theory to guide biological control of disease vectors. This necessitates a conceptual framework that explicitly considers a range of indirect interactions between the host-vector disease system and the vector's biological control agent. Here we conduct a comparative analysis of the efficacy of different types of biological control agents in controlling vector-borne diseases. We report three key findings. First, highly efficient predators and parasitoids of the vector prove to be effective biological control agents, but highly virulent pathogens of the vector also require a high transmission rate to be effective. Second, biocontrol agents can successfully reduce long-term host disease incidence even though they may fail to reduce long-term vector densities. Third, inundating a host-vector disease system with a natural enemy of the vector has little or no effect on reducing disease incidence, but inundating the system with a competitor of the vector has a large effect on reducing disease incidence. The comparative framework yields predictions that are useful in developing biological control strategies for vector-borne diseases. We discuss how these predictions can inform ongoing biological control efforts for host-vector disease systems. Copyright © 2012. Published by Elsevier Ltd.

  19. Improvement of the energy metabolism of recombinant CHO cells by cell sorting for reduced mitochondrial membrane potential.

    Science.gov (United States)

    Hinterkörner, Georg; Brugger, Gudrun; Müller, Dethardt; Hesse, Friedemann; Kunert, Renate; Katinger, Hermann; Borth, Nicole

    2007-05-10

    One of the major problems in process performance of mammalian cell cultures is the production of lactic acid. Cell specific glucose uptake rates usually correlate to glucose concentration and approximately 80% of the metabolised glucose is converted into lactic acid. As the mitochondrial membrane potential was shown to correlate to cell specific glucose uptake rates, we used Rhodamine 123, a lipophilic cationic dye for cell sorting to improve the energy metabolism of existing production cell lines. Two recombinant CHO cell lines with known differences in lactic acid production rate were used to evaluate Rhodamine 123 staining as a descriptor for glucose uptake rates and to determine whether it is possible to isolate subclones with altered metabolic properties. Such subclones would exhibit an improved process performance, and in addition could be used as models for genomic and metabolic studies. From the cell line with high lactate production, a subclone sorted for reduced mitochondrial membrane potential was found to have a lower specific lactate formation rate compared to the parental cell line in batch cultures. In addition, the glucose consumption rate was also reduced, while both the growth rate and the final cell concentration were increased. A subclone sorted for high mitochondrial membrane potential, on the other hand, had a higher glucose consumption rate, a higher lactate production rate and reduced growth. The potential of using flow cytometric cell sorting methods based on physiological activity for cell line optimisation is discussed.

  20. Origin and evolution of binucleated cells and binucleated cells with micronuclei in cisplatin-treated CHO cultures.

    Science.gov (United States)

    Rodilla, V

    1993-08-01

    It has recently been described that cisplatin is an agent able to induce binucleated cells (BC) in cultured CHO cells. Both the origin and the significance of those cells within a population are unknown although several hypothesis have been suggested such as blocking of cytokinesis or cell fusion. Using interval photography we have found that at least two mechanisms are involved in the production of BC. These cells can arise in a culture as a result of an incomplete process of cell division, i.e. karyokinesis with incomplete cytokinesis or as a result of the mitotic division of a pre-existent BC. The mitotic division of a BC can give rise to different types of daughter cells. These BC sometimes enter mitosis but fail to divide and as a consequence they remain BC. When the process of division is successful (in the vast majority of cases), the results that have been found are either two mononucleated cells or one mononucleated and one binucleated cell. The possible implications and significance of BC and BC with micronuclei in a given population are discussed.

  1. Photomutagenesis test development: II. 8-Methoxypsoralen, chlorpromazine and sunscreen compounds in chromosomal aberration assays using CHO cells.

    Science.gov (United States)

    Chételat, A; Dresp, J H; Gocke, E

    1993-12-01

    Chromosomal changes were analysed in Chinese hamster ovary (CHO) cells treated with 8-methoxypsoralen (8-MOP) or chlorpromazine (CPZ) and irradiated with either a UVA fluorescent tube (emission spectrum ranging from 350 to 400 nm) or a xenon burner (continuous emission spectrum simulating ambient sunlight). In the dark neither 8-MOP nor CPZ was genotoxic by itself. If these compounds were used in combination with UV irradiation the rate of chromosome aberrations was significantly increased. The magnitude of the clastogenic response was dependent on compound concentration and UV dose. The spectral composition also played an important role. Care must be taken to account for spectral changes caused, e.g., by passage of the light through the plastic lid of the container. The possible clastogenicity of two sunscreens was tested with two protocols: (1) cells attached to the culture dish were treated in presence of the sunscreen in the medium or (2) cells were irradiated through a layer of sunscreen solution as a filter. With this a clear UVB-absorbing effect and a decreased frequency of UVAB-induced chromosome aberration was evident with the UVB-absorbing compound Parsol HS but was absent, as expected, with the UVA-absorbing compound Parsol 1789. The presence of the sunscreens in the irradiated cell sample did not cause a significant increase in UV-induced chromosome aberrations.

  2. Production and glycosylation of recombinant beta-interferon in suspension and cytopore microcarrier cultures of CHO cells.

    Science.gov (United States)

    Spearman, Maureen; Rodriguez, Jose; Huzel, Norm; Butler, Michael

    2005-01-01

    Microcarriers are suitable for high-density cultures of cells requiring surface attachment and also offer the advantage of easy media removal for product recovery. We have used the macroporous microcarriers Cytopore 1 and 2 for the growth of CHO cells producing recombinant human beta-interferon (beta-IFN) in stirred batch cultures. Although these cells may grow in suspension, in the presence of Cytopore microcarriers they become entrapped in the inner bead matrix where they can be maintained at high densities. Cell growth rates were reduced in microcarrier cultures compared to suspension cultures. However, the beta-IFN yield was up to 3-fold greater as a result of an almost 5-fold higher specific productivity. Maximum productivity was found in cultures containing 1.0 mg/mL of Cytopore 1 or 0.5 mg/mL of Cytopore 2 with a cell/bead ratio of 1029 and 822, respectively. Beta-IFN molecules aggregated in the later stages of all cultures, causing a decrease in response by ELISA. However, the degree of aggregation was significantly less in the microcarrier cultures. The N-linked glycans from beta-IFN were isolated and analyzed by normal phase HPLC. There was no apparent difference in the profile of glycans obtained from each of the suspension and Cytopore culture systems. This suggests that Cytopore microcarriers may be useful in bioprocess development for enhanced recombinant glycoprotein production without affecting the glycosylation profile of the protein.

  3. Prevalence of Dementia and Mild Cognitive Impairment in the Rural Island Town of Ama-cho, Japan

    Directory of Open Access Journals (Sweden)

    Kenji Wada-Isoe

    2012-04-01

    Full Text Available Aims: In order to determine the prevalence of dementia and mild cognitive impairment (MCI, we conducted a population-based study in Japan. Methods: Participants included 924 subjects aged 65 years or older who resided in the town of Ama-cho. In phase 1 of the study, the Mini-Mental State Examination and Clinical Dementia Rating were administered for screening purposes. In phase 2 of the study, the subjects who screened positive were further examined by neurologists. Dementia and MCI were diagnosed by means of DSM-IV and International Working Group on MCI criteria, respectively. Results: By the prevalence date of June 1, 2010, 24 subjects had deceased or lived outside the town. In total, 723 of the remaining 900 subjects received a phase 1 test. In phase 2, 98 subjects were diagnosed with amnestic MCI, 113 subjects with non-amnestic MCI, and 82 subjects with dementia. Of the subjects who did not receive the phase 1 test, 66 subjects were diagnosed as having dementia according to data from their town medical card or the Long-term Care Insurance System. The crude prevalence of amnestic MCI, non-amnestic MCI, and dementia were 10.9, 12.6, and 16.4%, respectively. Conclusion: Consistent with the striking increase in the number of elderly individuals, we report higher prevalence of MCI and dementia in Japan than previously described.

  4. An omics approach to rational feed: Enhancing growth in CHO cultures with NMR metabolomics and 2D-DIGE proteomics.

    Science.gov (United States)

    Blondeel, Eric J M; Ho, Raymond; Schulze, Steffen; Sokolenko, Stanislav; Guillemette, Simon R; Slivac, Igor; Durocher, Yves; Guillemette, J Guy; McConkey, Brendan J; Chang, David; Aucoin, Marc G

    2016-09-20

    Expression of recombinant proteins exerts stress on cell culture systems, affecting the expression of endogenous proteins, and contributing to the depletion of nutrients and accumulation of waste metabolites. In this work, 2D-DIGE proteomics was employed to analyze differential expression of proteins following stable transfection of a Chinese Hamster Ovary (CHO) cell line to constitutively express a heavy-chain monoclonal antibody. Thirty-four proteins of significant differential expression were identified and cross-referenced with cellular functions and metabolic pathways to identify points of cell stress. Subsequently, 1D-(1)H NMR metabolomics experiments analyzed cultures to observe nutrient depletion and waste metabolite accumulations to further examine these cell stresses and pathways. From among fifty metabolites tracked in time-course, eight were observed to be completely depleted from the production media, including: glucose, glutamine, proline, serine, cystine, asparagine, choline, and hypoxanthine, while twenty-three excreted metabolites were also observed to accumulate. The differentially expressed proteins, as well as the nutrient depletion and accumulation of these metabolites corresponded with upregulated pathways and cell systems related to anaplerotic TCA-replenishment, NADH/NADPH replenishment, tetrahydrofolate cycle C1 cofactor conversions, limitations to lipid synthesis, and redox modulation. A nutrient cocktail was assembled to improve the growth medium and alleviate these cell stresses to achieve a ∼75% improvement to peak cell densities. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Isotope labeling to determine the dynamics of metabolic response in CHO cell perfusion bioreactors using MALDI-TOF-MS.

    Science.gov (United States)

    Karst, Daniel J; Steinhoff, Robert F; Kopp, Marie R G; Soos, Miroslav; Zenobi, Renato; Morbidelli, Massimo

    2017-08-25

    The steady-state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI-TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP-Hex, UDP-HexNAc). By switching to a (13) C6 glucose containing feed media during constant operation at 20 × 10(6) cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the (13) C6 glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools, τST (≤0.56 days), which were confirmed in the bioreactor. On the other hand, it is shown that the reactor residence time τR (1 day) and characteristic time for glucose uptake τGlc (0.33 days), representative of the bioreactor dynamics, delayed the consumption of (13) C6 glucose in the bioreactor and thus the intracellular (13) C enrichment. The proposed experimental approach allowed the decoupling of bioreactor hydrodynamics and intrinsic dynamics of cell metabolism in response to a change in the cell culture environment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

  6. Isolation and characterization of Chinese hamster ovary (CHO) cells deficient in acyl coenzyme A: cholesterol acyltransferase (ACAT) activity

    Energy Technology Data Exchange (ETDEWEB)

    Cadigan, K.M.; Heider, J.G.; Chang, T.Y.

    1986-05-01

    The specific ACAT inhibitor compound 58-035 has been used to mimic the phenotype of an ACAT deficient mutant in 25-RA cells. 25-RA is a CHO cell line resistant to 25-hydroxycholesterol and contains five times more cholesterol ester than wild-type (WT) cells. 25-RA cells preincubated with 58-035 are 100 to 500 times more resistant to amphotericin B killing than untreated 25-RA. 100 x 10/sup 6/ mutagenized 25-RA cells underwent three rounds of amphotericin B killing and two rounds of 25-hydroxycholesterol killing (to remove WT revertants which are amphotericin B resistant). Thus far, three biochemically distinct mutants have been isolated containing 33% (AC27), 25% (AC90), and 10% (AC232) of the parental ACAT activity as measured by an /sup 3/H-oleate pulse in intact cells. When parental and mutant cell extracts are reconstituted into cholesterol containing liposomes the differences in ACAT activity remain. They have also found that 25-RA cells can survive in cholesterol free medium containing TMD, an inhibitor of cholesterol biosynthesis, presumably because of adequate supply of endogenous cholesterol from hydrolysis of its stored cholesterol ester. In contrast, under the same conditions, mutant AC232 is effectively killed ( greater than or equal to 99%) by cholesterol starvation, thus providing a potential selection procedure for isolating revertants of ACAT mutants.

  7. Zika Virus Mosquito Vectors: Competence, Biology, and Vector Control.

    Science.gov (United States)

    Kauffman, Elizabeth B; Kramer, Laura D

    2017-12-16

    Zika virus (ZIKV) (Flaviviridae, Flavivirus) has become one of the most medically important mosquito-borne viruses because of its ability to cause microcephaly in utero and Guillain-Barré syndrome in adults. This virus emerged from its sylvatic cycle in Africa to cause an outbreak in Yap, Federated States of Micronesia in 2007, French Polynesia in 2014, and most recently South America in 2015. The rapid expansion of ZIKV in the Americas largely has been due to the biology and behavior of its vector, Aedes aegypti. Other arboviruses transmitted by Ae. aegypti include the 2 flaviviruses dengue virus and yellow fever virus and the alphavirus chikungunya virus, which are also (re)emerging viruses in the Americas. This mosquito vector is highly domesticated, living in close association with humans in urban households. Its eggs are desiccation resistant, and the larvae develop rapidly in subtropical and tropical environments. Climate warming is facilitating range expansion of Ae. aegypti, adding to the threat this mosquito poses to human health, especially in light of the difficulty controlling it. Aedes albopictus, another highly invasive arbovirus vector that has only been implicated in one country (Gabon), is an important vector of ZIKV, but because of its wide geographic distribution may become a more important vector in the future. This article discusses the historical background of ZIKV and the biology and ecology of these 2 vectors. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  8. Cytoskeleton, endoplasmic reticulum and nucleus alterations in CHO-K1 cell line after Crotalus durissus terrificus (South American rattlesnake venom treatment

    Directory of Open Access Journals (Sweden)

    B. P. Tamieti

    2007-01-01

    Full Text Available Snake venoms are toxic to a variety of cell types. However, the intracellular damages and the cell death fate induced by venom are unclear. In the present work, the action of the South American rattlesnake Crotalus durissus terrificus venom on CHO-K1 cell line was analyzed. The cells CHO-K1 were incubated with C. d. terrificus venom (10, 50 and 100g/ml for 1 and 24 hours, and structural alterations of actin filaments, endoplasmic reticulum and nucleus were assessed using specific fluorescent probes and agarose gel electrophoresis for DNA fragmentation. Significant structural changes were observed in all analyzed structures. DNA fragmentation was detected suggesting that, at the concentrations used, the venom induced apoptosis.

  9. A study on the management solution by a local resident group in historical building case study in Ikuno-cho, Asago City

    Science.gov (United States)

    Tajima, Kimie

    2017-10-01

    The purpose of this research is to provide some useful knowledge for future operation and preservation of historical buildings through the field survey about how one of the historical buildings is being managed and preserved by local residents. This study surveyed one historical building, Izutsu-ya, located in Ikuno-cho, Asago City. The reasons why this study focuses on this building are the following: First, Izutsu-ya is the first building managed and preserved by local residents in Asago City; second, Ikuno-cho is one of the well-known places where community development is being performed. The following three methods were used in this study: hearing survey, behavior observation survey, and questionnaire survey. As a result, this research mainly showed that local residents should be involved in the maintenance and preservation from the early stage of the activity, and old buildings should be utilized for a larger variety of purposes.

  10. Generalized Selection Weighted Vector Filters

    Directory of Open Access Journals (Sweden)

    Rastislav Lukac

    2004-09-01

    Full Text Available This paper introduces a class of nonlinear multichannel filters capable of removing impulsive noise in color images. The here-proposed generalized selection weighted vector filter class constitutes a powerful filtering framework for multichannel signal processing. Previously defined multichannel filters such as vector median filter, basic vector directional filter, directional-distance filter, weighted vector median filters, and weighted vector directional filters are treated from a global viewpoint using the proposed framework. Robust order-statistic concepts and increased degree of freedom in filter design make the proposed method attractive for a variety of applications. Introduced multichannel sigmoidal adaptation of the filter parameters and its modifications allow to accommodate the filter parameters to varying signal and noise statistics. Simulation studies reported in this paper indicate that the proposed filter class is computationally attractive, yields excellent performance, and is able to preserve fine details and color information while efficiently suppressing impulsive noise. This paper is an extended version of the paper by Lukac et al. presented at the 2003 IEEE-EURASIP Workshop on Nonlinear Signal and Image Processing (NSIP '03 in Grado, Italy.

  11. A generalized nonlocal vector calculus

    Science.gov (United States)

    Alali, Bacim; Liu, Kuo; Gunzburger, Max

    2015-10-01

    A nonlocal vector calculus was introduced in Du et al. (Math Model Meth Appl Sci 23:493-540, 2013) that has proved useful for the analysis of the peridynamics model of nonlocal mechanics and nonlocal diffusion models. A formulation is developed that provides a more general setting for the nonlocal vector calculus that is independent of particular nonlocal models. It is shown that general nonlocal calculus operators are integral operators with specific integral kernels. General nonlocal calculus properties are developed, including nonlocal integration by parts formula and Green's identities. The nonlocal vector calculus introduced in Du et al. (Math Model Meth Appl Sci 23:493-540, 2013) is shown to be recoverable from the general formulation as a special example. This special nonlocal vector calculus is used to reformulate the peridynamics equation of motion in terms of the nonlocal gradient operator and its adjoint. A new example of nonlocal vector calculus operators is introduced, which shows the potential use of the general formulation for general nonlocal models.

  12. Taguchi Experimental Design for Optimization of Recombinant Human Growth Hormone Production in CHO Cell Lines and Comparing its Biological Activity with Prokaryotic Growth Hormone.

    Science.gov (United States)

    Aghili, Zahra Sadat; Zarkesh-Esfahani, Sayyed Hamid

    2018-02-01

    Growth hormone deficiency results in growth retardation in children and the GH deficiency syndrome in adults and they need to receive recombinant-GH in order to rectify the GH deficiency symptoms. Mammalian cells have become the favorite system for production of recombinant proteins for clinical application compared to prokaryotic systems because of their capability for appropriate protein folding, assembly, post-translational modification and proper signal. However, production level in mammalian cells is generally low compared to prokaryotic hosts. Taguchi has established orthogonal arrays to describe a large number of experimental situations mainly to reduce experimental errors and to enhance the efficiency and reproducibility of laboratory experiments.In the present study, rhGH was produced in CHO cells and production of rhGH was assessed using Dot blotting, western blotting and Elisa assay. For optimization of rhGH production in CHO cells using Taguchi method An M16 orthogonal experimental design was used to investigate four different culture components. The biological activity of rhGH was assessed using LHRE-TK-Luciferase reporter gene system in HEK-293 and compared to the biological activity of prokaryotic rhGH.A maximal productivity of rhGH was reached in the conditions of 1%DMSO, 1%glycerol, 25 µM ZnSO 4 and 0 mM NaBu. Our findings indicate that control of culture conditions such as the addition of chemical components helps to develop an efficient large-scale and industrial process for the production of rhGH in CHO cells. Results of bioassay indicated that rhGH produced by CHO cells is able to induce GH-mediated intracellular cell signaling and showed higher bioactivity when compared to prokaryotic GH at the same concentrations. © Georg Thieme Verlag KG Stuttgart · New York.

  13. Promotion of Cholera Awareness Among Households of Cholera Patients: A Randomized Controlled Trial of the Cholera-Hospital-Based-Intervention-for-7 Days (CHoBI7) Intervention.

    Science.gov (United States)

    Saif-Ur-Rahman, K M; Parvin, Tahmina; Bhuyian, Sazzadul Islam; Zohura, Fatema; Begum, Farzana; Rashid, Mahamud-Ur; Biswas, Shwapon Kumar; Sack, David; Sack, R Bradley; Monira, Shirajum; Alam, Munirul; Shaly, Nusrat Jahan; George, Christine Marie

    2016-12-07

    Previous studies have demonstrated that household contacts of cholera patients are highly susceptible to cholera infections for a 7-day period after the presentation of the index patient in the hospital. However, there is no standard of care to prevent cholera transmission in this high-risk population. Furthermore, there is limited information available on awareness of cholera transmission and prevention among cholera patients and their household contacts. To initiate a standard of care for this high-risk population, we developed the Cholera-Hospital-Based-Intervention-for-7-Days (CHoBI7), which delivers a handwashing with soap and water treatment intervention to household contacts during the time they spend with the admitted cholera patient in the hospital and reinforces these messages through home visits. To test CHoBI7, we conducted a randomized controlled trial among 302 intervention cholera patient household members and 302 control cholera patient household members in Dhaka, Bangladesh. In this study, we evaluated the effectiveness of the CHoBI7 intervention in increasing awareness of cholera transmission and prevention, and the key times for handwashing with soap. We observed a significant increase in cholera knowledge score in the intervention arm compared with the control arm at both the 1-week follow-up {score coefficient = 2.34 (95% confidence interval [CI] = 1.96, 2.71)} and 6 to 12-month follow-up period (score coefficient = 1.59 [95% CI = 1.05, 2.13]). This 1-week hospital- and home-based intervention led to a significant increase in knowledge of cholera transmission and prevention which was sustained 6 to 12 months post-intervention. These findings suggest that the CHoBI7 intervention presents a promising approach to increase cholera awareness among this high-risk population. © The American Society of Tropical Medicine and Hygiene.

  14. Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

    Energy Technology Data Exchange (ETDEWEB)

    Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Fujita, Norihisa, E-mail: nori@ph.ritsumei.ac.jp [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); School of Pharmacy, Ristumeikan University, Kusatsu, Shiga 525-8577 (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. Black-Right-Pointing-Pointer These are due to increase in intracellular cAMP concentration. Black-Right-Pointing-Pointer Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. Black-Right-Pointing-Pointer The potencies of fatty acid moiety on LysoPS were oleoyl Greater-Than-Or-Slanted-Equal-To stearoyl > palmitoyl. Black-Right-Pointing-Pointer We propose that GPR174 is a lysophosphatidylserine receptor. -- Abstract: We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of G{alpha}{sub s} protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G{alpha}{sub s}, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

  15. The Effect of Leonurus sibiricus Plant Extracts on Stimulating Repair and Protective Activity against Oxidative DNA Damage in CHO Cells and Content of Phenolic Compounds.

    Science.gov (United States)

    Sitarek, Przemysław; Skała, Ewa; Wysokińska, Halina; Wielanek, Marzena; Szemraj, Janusz; Toma, Monika; Śliwiński, Tomasz

    2016-01-01

    Leonurus sibiricus L. has been used as a traditional and medicinal herb for many years in Asia and Europe. This species is known to have antibacterial, anti-inflammatory, and antioxidant activity and has demonstrated a reduction of intracellular reactive oxygen species. All tested extracts of L. sibiricus showed protective and DNA repair stimulating effects in Chinese hamster ovary (CHO) cells exposed to H2O2. Preincubation of the CHO cells with 0.5 mg/mL of plant extracts showed increased expression level of antioxidant genes (SOD2, CAT, and GPx). LC-MS/MS and HPLC analyses revealed the presence of nine phenolic compounds in L. sibiricus plant extracts: catechin, verbascoside, two flavonoids (quercetin and rutin), and five phenolic acids (4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid). The roots and aerial parts of in vitro L. sibiricus plant extracts, which had the strongest antioxidant properties, may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, as well as protecting DNA via enhanced activation of the antioxidant genes (SOD2, CAT, and GPx) regulating intracellular antioxidant capacity. The content of phenolic compounds in in vitro raised plants was greater than the levels found in plants propagated from seeds.

  16. The Effect of Leonurus sibiricus Plant Extracts on Stimulating Repair and Protective Activity against Oxidative DNA Damage in CHO Cells and Content of Phenolic Compounds

    Directory of Open Access Journals (Sweden)

    Przemysław Sitarek

    2016-01-01

    Full Text Available Leonurus sibiricus L. has been used as a traditional and medicinal herb for many years in Asia and Europe. This species is known to have antibacterial, anti-inflammatory, and antioxidant activity and has demonstrated a reduction of intracellular reactive oxygen species. All tested extracts of L. sibiricus showed protective and DNA repair stimulating effects in Chinese hamster ovary (CHO cells exposed to H2O2. Preincubation of the CHO cells with 0.5 mg/mL of plant extracts showed increased expression level of antioxidant genes (SOD2, CAT, and GPx. LC-MS/MS and HPLC analyses revealed the presence of nine phenolic compounds in L. sibiricus plant extracts: catechin, verbascoside, two flavonoids (quercetin and rutin, and five phenolic acids (4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid. The roots and aerial parts of in vitro L. sibiricus plant extracts, which had the strongest antioxidant properties, may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, as well as protecting DNA via enhanced activation of the antioxidant genes (SOD2, CAT, and GPx regulating intracellular antioxidant capacity. The content of phenolic compounds in in vitro raised plants was greater than the levels found in plants propagated from seeds.

  17. A Single Dynamic Metabolic Model Can Describe mAb Producing CHO Cell Batch and Fed-Batch Cultures on Different Culture Media.

    Science.gov (United States)

    Robitaille, Julien; Chen, Jingkui; Jolicoeur, Mario

    2015-01-01

    CHO cell culture high productivity relies on optimized culture medium management under fed-batch or perfused chemostat strategies enabling high cell densities. In this work, a dynamic metabolic model for CHO cells was further developed, calibrated and challenged using datasets obtained under four different culture conditions, including two batch and two fed-batch cultures comparing two different culture media. The recombinant CHO-DXB11 cell line producing the EG2-hFc monoclonal antibody was studied. Quantification of extracellular substrates and metabolites concentration, viable cell density, monoclonal antibody concentration and intracellular concentration of metabolite intermediates of glycolysis, pentose-phosphate and TCA cycle, as well as of energetic nucleotides, were obtained for model calibration. Results suggest that a single model structure with a single set of kinetic parameter values is efficient at simulating viable cell behavior in all cases under study, estimating the time course of measured and non-measured intracellular and extracellular metabolites. Model simulations also allowed performing dynamic metabolic flux analysis, showing that the culture media and the fed-batch strategies tested had little impact on flux distribution. This work thus paves the way to an in silico platform allowing to assess the performance of different culture media and fed-batch strategies.

  18. Synthetic Aperture Vector Flow Imaging

    DEFF Research Database (Denmark)

    Oddershede, Niels

    2008-01-01

    Current ultrasonic blood flow velocity measurement systems are subject to a number of limitations, including limited frame rate, aliasing artifacts, and that only the velocity component along the ultrasound beam is estimated. This dissertation aims at solving some of these problems. The main part...... of the thesis considers a method for estimating the two-dimensional velocity vector within the image plane. This method, called synthetic aperture vector flow imaging, is first shortly reviewed. The main contribution of this work is partly an analysis of the method with respect to focusing effects, motion...... estimation. The method can be used for increasing the frame rate of color flow maps or alternatively for a new imaging modality entitled quadroplex imaging, featuring a color flow map and two independent spectrograms at a high frame rate. The second is an alternative method for ultrasonic vector velocity...

  19. Gauge Theories of Vector Particles

    Science.gov (United States)

    Glashow, S. L.; Gell-Mann, M.

    1961-04-24

    The possibility of generalizing the Yang-Mills trick is examined. Thus we seek theories of vector bosons invariant under continuous groups of coordinate-dependent linear transformations. All such theories may be expressed as superpositions of certain "simple" theories; we show that each "simple theory is associated with a simple Lie algebra. We may introduce mass terms for the vector bosons at the price of destroying the gauge-invariance for coordinate-dependent gauge functions. The theories corresponding to three particular simple Lie algebras - those which admit precisely two commuting quantum numbers - are examined in some detail as examples. One of them might play a role in the physics of the strong interactions if there is an underlying super-symmetry, transcending charge independence, that is badly broken. The intermediate vector boson theory of weak interactions is discussed also. The so-called "schizon" model cannot be made to conform to the requirements of partial gauge-invariance.

  20. 3-D Vector Flow Imaging

    DEFF Research Database (Denmark)

    Holbek, Simon

    ultrasonic vector flow estimation and bring it a step closer to a clinical application. A method for high frame rate 3-D vector flow estimation in a plane using the transverse oscillation method combined with a 1024 channel 2-D matrix array is presented. The proposed method is validated both through phantom......For the last decade, the field of ultrasonic vector flow imaging has gotten an increasingly attention, as the technique offers a variety of new applications for screening and diagnostics of cardiovascular pathologies. The main purpose of this PhD project was therefore to advance the field of 3-D...... hampers the task of real-time processing. In a second study, some of the issue with the 2-D matrix array are solved by introducing a 2-D row-column (RC) addressing array with only 62 + 62 elements. It is investigated both through simulations and via experimental setups in various flow conditions...

  1. Toward lattice fractional vector calculus

    Science.gov (United States)

    Tarasov, Vasily E.

    2014-09-01

    An analog of fractional vector calculus for physical lattice models is suggested. We use an approach based on the models of three-dimensional lattices with long-range inter-particle interactions. The lattice analogs of fractional partial derivatives are represented by kernels of lattice long-range interactions, where the Fourier series transformations of these kernels have a power-law form with respect to wave vector components. In the continuum limit, these lattice partial derivatives give derivatives of non-integer order with respect to coordinates. In the three-dimensional description of the non-local continuum, the fractional differential operators have the form of fractional partial derivatives of the Riesz type. As examples of the applications of the suggested lattice fractional vector calculus, we give lattice models with long-range interactions for the fractional Maxwell equations of non-local continuous media and for the fractional generalization of the Mindlin and Aifantis continuum models of gradient elasticity.

  2. Influence of the host (Cho) and of the cultivation strategy on glycan structures and molecular properties of human thyrotrophin; Influencia do hospedeiro (Cho) e da estrategia de cultivo nas estruturas glicidicas e propriedades moleculares da tireotrofina humana

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, Joao Ezequiel de

    2007-07-01

    A novel, fast and practical two-step purification strategy, consisting of a classical ion exchange and a reversed-phase high performance liquid chromatography (RP-HPLC), for rapidly obtaining CHO-derived hTSH, was set up providing r-hTSH with 70% yield and > 99% purity. A consistent increase of {approx} 60% in the secretion yields of r-hTSH-IPEN was observed by changing cell culture CO{sub 2} conditions from 5% CO{sub 2} to air environment (0.03% CO{sub 2}). The overall quality of the products obtained under both conditions was evaluated for what concerns N-glycan structure, charge isomers and biological activity in comparison with a well known recombinant biopharmaceutical (Thyrogen{sup R}) and with a pituitary reference preparation (p-hTSH) from National Hormone and Pituitary Program (NIDDK, USA). The N-glycans identified in the recombinant preparations were of the complex type, presenting bi-, tri- and tetra-antennary structures, sometimes fucosylated, 86-88% of the identified structures being sialylated at variable levels. The three most abundant structures were monosialylated glycans, representing {approx} 69% of all identified forms in the three preparations. The main difference was found in terms of antennarity, with 8-10% more bi-antennary structures obtained in the absence of CO{sub 2} and 7-9% more tri-antennary structures in its presence. In the case of p-hTSH, complex, high-mannose and hybrid N-glycan structures were identified, most of them containing sialic acid and/or sulphate terminal residues. The two most abundant structures were shown to contain one or two sulphate residues, one of which unexpectedly bound to galactose. The sialic acid-galactose linkage was also determined, having found that 68 3 {+-} 10% was in the {alpha} 2,6 and 32 {+-} 10% in the {alpha}2,3 conformation. No remarkable difference in charge isomers was observed between the three recombinant preparations, the isoelectric focusing profiles showing six distinct bands in the 5

  3. Learning with Support Vector Machines

    CERN Document Server

    Campbell, Colin

    2010-01-01

    Support Vectors Machines have become a well established tool within machine learning. They work well in practice and have now been used across a wide range of applications from recognizing hand-written digits, to face identification, text categorisation, bioinformatics, and database marketing. In this book we give an introductory overview of this subject. We start with a simple Support Vector Machine for performing binary classification before considering multi-class classification and learning in the presence of noise. We show that this framework can be extended to many other scenarios such a

  4. Topological vector spaces and distributions

    CERN Document Server

    Horvath, John

    2012-01-01

    ""The most readable introduction to the theory of vector spaces available in English and possibly any other language.""-J. L. B. Cooper, MathSciNet ReviewMathematically rigorous but user-friendly, this classic treatise discusses major modern contributions to the field of topological vector spaces. The self-contained treatment includes complete proofs for all necessary results from algebra and topology. Suitable for undergraduate mathematics majors with a background in advanced calculus, this volume will also assist professional mathematicians, physicists, and engineers.The precise exposition o

  5. VECTOR MAPS IN MOBILE ROBOTICS

    Directory of Open Access Journals (Sweden)

    Ales Jelinek

    2015-12-01

    Full Text Available The aim of this paper is to provide a brief overview of vector map techniques used in mobile robotics and to present current state of the research in this field at the Brno University of Technology. Vector maps are described as a part of the simultaneous localization and mapping (SLAM problem in the environment without artificial landmarks or global navigation system. The paper describes algorithms from data acquisition to map building but particular emphasis is put on segmentation, line extraction and scan matching algorithms. All significant algorithms are illustrated with experimental results.

  6. Cascading effect in bioprocessing-The impact of mild hypothermia on CHO cell behavior and host cell protein composition.

    Science.gov (United States)

    Goey, Cher H; Tsang, Joshua M H; Bell, David; Kontoravdi, Cleo

    2017-12-01

    A major challenge in downstream purification of monoclonal antibodies (mAb) is the removal of host cell proteins (HCPs). Previous studies have shown that cell culture conditions significantly impact the HCP content at harvest. However, it is currently unclear how process conditions affect physiological changes in the host cell population, and how these changes, in turn, cascade down to change the HCP profile. We examined how temperature downshift (TDS) to mild hypothermia affects key upstream performance indicators, that is antibody titre, HCP concentration and HCP species, across the cell culture decline phase and at harvest through the lens of changes in cellular behavior. Mild hypothermic conditions introduced on day 5 of fed-batch Chinese hamster ovary (CHO) cell bioreactors resulted in a lower cell proliferation rate but larger percentages of healthier cells across the cell culture decline phase compared to bioreactors maintained at standard physiological temperature. Moreover, the onset of apoptosis was less evident in mild hypothermic cultures. Consequently, mild hypothermic cultures took an extra 5 days to reach an integral viable cell concentration (IVCC) and antibody yield similar to that of the control at standard physiological temperature. When cell viability dropped below 80%, mild hypothermic cell cultures had a reduced variety of HCP species by 36%, including approximately 44% and 27% lower proteases and chaperones, respectively, despite having similar HCP concentration. This study suggests that TDS may be a good strategy to provide cleaner downstream feedstocks by reducing the variety of HCPs and to maintain product integrity by reducing the number of proteases and chaperones. © 2017 Wiley Periodicals, Inc.

  7. Investigation of superparamagnetic (Fe3O4) nanoparticles and magnetic field exposures on CHO-K1 cell line

    Science.gov (United States)

    Coker, Zachary; Estlack, Larry; Hussain, Saber; Choi, Tae-Youl; Ibey, Bennett L.

    2016-03-01

    Rapid development in nanomaterial synthesis and functionalization has led to advanced studies in actuation and manipulation of cellular functions for biomedical applications. Often these actuation techniques employ externally applied magnetic fields to manipulate magnetic nanomaterials inside cell bodies in order to drive or trigger desired effects. While cellular interactions with low-frequency magnetic fields and nanoparticles have been extensively studied, the fundamental mechanisms behind these interactions remain poorly understood. Additionally, modern investigations on these concurrent exposure conditions have been limited in scope, and difficult to reproduce. This study presents an easily reproducible method of investigating the biological impact of concurrent magnetic field and nanoparticle exposure conditions using an in-vitro CHO-K1 cell line model, with the purpose of establishing grounds for in-depth fundamental studies of the mechanisms driving cellular-level interactions. Cells were cultured under various nanoparticle and magnetic field exposure conditions from 0 to 500 μg/ml nanoparticle concentrations, and DC, 50 Hz, or 100 Hz magnetic fields with 2.0 mT flux density. Cells were then observed by confocal fluorescence microscopy, and subject to biological assays to determine the effects of concurrent extreme-low frequency magnetic field and nanoparticle exposures on cellnanoparticle interactions, such as particle uptake and cell viability by MTT assay. Current results indicate little to no variation in effect on cell cultures based on magnetic field parameters alone; however, it is clear that deleterious synergistic effects of concurrent exposure conditions exist based on a significant decrease in cell viability when exposed to high concentrations of nanoparticles and concurrent magnetic field.

  8. Effects of lunar and mars dust on HaCaT keratinocytes and CHO-K1 fibroblasts

    Science.gov (United States)

    Brix, Klaudia; Slenzka, Klaus; Rehders, Maren; Sadhukhan, Annapurna; Mistry, Rima; Duenne, Matthias; Kempf, Juergen

    Exposure to lunar dust during Apollo missions resulted in occasional reports of ocular, respira-tory and dermal irritations which showed that lunar dust has a risk potential for human health. This is caused by its high reactivity as well as its small size, leading to a wide distribution also inside habitats. Hence, detailed information regarding effects of lunar dust on human health is required to best support future missions to moon. In this study, we used different methods to assess the specific effects of lunar dust onto mammalian skin by exposing HaCaT keratinocytes and CHO-K1 fibroblasts to dusts simulating lunar or mars soils. These particular cell types were chosen because the skin protects the human body from potentially harmful substances and since a well orchestrated program ensures proper repair in cases of wounding. Keratinocytes and fibroblasts were exposed to the dusts for different durations of time and their effects on morphology, metabolic state, survival and proliferation of the cells were determined. Cytotoxi-city and proliferation were measured using the MTT assay, metabolic activity was analyzed by vital staining of mitochondria, and phalloidin staining of the actin cytoskeleton was performed to address structural integrity of the cells. It was found that the effects of the two types of soils on the different features of both cell lines varied to considerable extent, and that lunar and mars dust were specific in their effects. The obtained results will facilitate detailed inves-tigations of dust exposure during wound healing and will ease risk assessment studies for e.g. lunar lander approaches. The investigations will help to assess the risks and to determine safety measures to be taken during extraterrestrial expeditions in order to minimize risks to human health associated with exposure of human skin to dust contaminants.

  9. Effects of lunar and mars dust simulants on HaCaT keratinocytes and CHO-K1 fibroblasts

    Science.gov (United States)

    Rehders, Maren; Grosshäuser, Bianka B.; Smarandache, Anita; Sadhukhan, Annapurna; Mirastschijski, Ursula; Kempf, Jürgen; Dünne, Matthias; Slenzka, Klaus; Brix, Klaudia

    2011-04-01

    Exposure to lunar dust during Apollo missions resulted in occasional reports of ocular, respiratory and dermal irritations which showed that lunar dust has a risk potential for human health. This is caused by its high reactivity as well as its small size, leading to a wide distribution also inside habitats. Hence, detailed information regarding effects of extraterrestrial lunar dusts on human health is required to best support future missions to moon, mars or other destinations. In this study, we used several methods to assess the specific effects of extraterrestrial dusts onto mammalian skin by exposing HaCaT keratinocytes and CHO-K1 fibroblasts to dusts simulating lunar or mars soils. These particular cell types were chosen because the skin protects the human body from potentially harmful substances and because a well orchestrated program ensures proper wound healing. Keratinocytes and fibroblasts were exposed to the dusts for different durations of time and their effects on morphology and viability of the cells were determined. Cytotoxicity was measured using the MTT assay and by monitoring culture impedance, while phalloidin staining of the actin cytoskeleton was performed to address structural integrity of the cells which was also investigated by propidium iodide intake. It was found that the effects of the two types of dust simulants on the different features of both cell lines varied to a considerable extent. Moreover, proliferation of HaCaT keratinocytes, as analyzed by Ki67 labeling, was suppressed in sub-confluent cultures exposed to lunar dust simulant. Furthermore, experimental evidence is provided for a delay in regeneration of keratinocyte monolayers from scratch-wounding when exposed to lunar dust simulant. The obtained results will facilitate further investigations of dust exposure during wound healing and will ease risk assessment studies e.g., for lunar lander approaches. The investigations will help to determine safety measures to be taken during

  10. CHO-K1 host cells adapted to growth in glutamine-free medium by FACS-assisted evolution.

    Science.gov (United States)

    Bort, Juan A Hernández; Stern, Beate; Borth, Nicole

    2010-10-01

    During the process of recombinant cell line optimisation for production of biopharmaceuticals, multiple cellular properties like robustness against stress, the attainment of high cell concentrations and maintenance of high viability must be considered to maximize protein yield. To improve growth and viability, glutamine is supplemented as an alternative energy source for rapidly dividing cells that oxidize glucose inefficiently. However, the resulting by-product ammonia is toxic at high concentrations and has a negative impact on protein glycosylation, a major quality-determining parameter of biopharmaceuticals. In this work, the CHO-K1 cell line was adapted to a chemically defined medium and suspension growth within 3 weeks. Subsequently, the glutamine concentration was stepwise reduced from 8 to 4 and 2 mM. After each reduction, both the final cell concentration in the batch and the viability decreased. To force a rapid evolution of cells to achieve high final cell concentrations, cells were seeded at high densities (10(7) cells/mL) and surviving cells were sorted by FACS or MACS when viability declined to 10% (typically after 24 h). Sorted cells were grown in batch until viability declined to 10% and viable cells recovered again. The final sorted population was able to reach comparable or even better viable cell concentrations and showed a significantly improved viability compared to their ancestors. The 2 mM glutamine-adapted cell line was directly transferred into glutamine-free medium and was able to grow at comparable rates without requiring further adaptation. Cells compensated the lack of glutamine by increasing their consumption of glutamate and aspartate.

  11. Intracellular response to process optimization and impact on productivity and product aggregates for a high-titer CHO cell process.

    Science.gov (United States)

    Handlogten, Michael W; Lee-O'Brien, Allison; Roy, Gargi; Levitskaya, Sophia V; Venkat, Raghavan; Singh, Shailendra; Ahuja, Sanjeev

    2018-01-01

    A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentration, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression, and heavy and light chain mRNA expression provided an in-depth understanding of the cellular response to process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra- and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally

  12. SCALAR AND VECTOR IN COMPULATION

    Directory of Open Access Journals (Sweden)

    Valery F. Ochkov

    2013-01-01

    Full Text Available The article deals with two fundamental data types – scalar and vector (array, without the ability of working with them one cannot solve using computer school or university tasks in mathematics, physics, chemistry and other technical training courses. Some fundamentals of teaching computer science at school and university are covered as well. 

  13. Vector description of nonlinear magnetization

    Energy Technology Data Exchange (ETDEWEB)

    Rysak, A. E-mail: rysak@tytan.umcs.lublin.pl; Korczak, S.Z

    2001-06-01

    The definition of the nonlinear magnetization, which is usually applied in investigating superconductors is discussed in this paper. The nonlinear magnetization of the superconductor, as the response to the alternating magnetic field, is a periodic time function and is determined by the sequence of complex numbers {chi}{sub n}={chi}{sub n}'+i{chi}{sub n}'', n=1,2,3,.... The sequence {chi}{sub n} is named harmonic susceptibility. Finding some limits in this definition we propose a new description, substituting the complex sequence {chi}{sub n} with the sequence of real covariant vectors. Such a definition of the nonlinear magnetization, allows to determine function M(t) of an investigated system for an arbitrary initial phase {theta}{sub o} of the inducing magnetic field H{sub ac}=h{sub ac} sin({omega}t+{theta}{sub o}). Vector description, in our opinion, is superior to the complex one in respect of explicitness, simplicity, and universality. Circular diagrams are used to illustrate the new vector description. We also show how the vectors of the harmonic susceptibility can be derived from both numeric calculations and experimental data.

  14. Disease Vector Ecology Profile: Ecuador

    Science.gov (United States)

    1998-12-01

    species) only during the pupal and adult stages. Formulations of Bacillus thuringiensis israelensis are economical, operationally feasible, and...12. Bees , Wasps and Hornets ..................................................................................31 B. Snakes...epidemic proportions. Transmission of the plague bacillus by fleas is species- specific. Not all fleas are competent vectors. Xenopsylla cheopis is the

  15. Parallel Sparse Matrix - Vector Product

    DEFF Research Database (Denmark)

    Alexandersen, Joe; Lazarov, Boyan Stefanov; Dammann, Bernd

    This technical report contains a case study of a sparse matrix-vector product routine, implemented for parallel execution on a compute cluster with both pure MPI and hybrid MPI-OpenMP solutions. C++ classes for sparse data types were developed and the report shows how these class can be used...

  16. Synthetic Aperture Vector Flow Imaging

    DEFF Research Database (Denmark)

    Villagómez Hoyos, Carlos Armando

    The main objective of this project was to continue the development of a synthetic aperture vector flow estimator. This type of estimator is capable of overcoming two of the major limitations in conventional ultrasound systems: 1) the inability to scan large region of interest with high temporal r...

  17. The consequences of poor vectorization

    CERN Multimedia

    CERN. Geneva

    2016-01-01

    This talk briefly discusses the vectorization problem and how it impacts scientific and engineering systems. A simple cost model of designing such system in context of different phases of software lifetime is considered. Finally a concept for scalable solution is presented.

  18. Constraints in vector meson photoproduction

    Energy Technology Data Exchange (ETDEWEB)

    Kloet, W. M.; Tabakin, F

    2000-01-31

    Constraints are discussed for spin observables extracted from photoproduction of vector mesons. These constraints originate from positivity of the spin density matrix and should be part of any future analysis of experimental data. Spin observables need to be defined in the photon-nucleon c.m. frame.

  19. Constraints in Vector Meson Photoproduction

    Science.gov (United States)

    Kloet, W. M.; Tabakin, F.

    2000-01-01

    Constraints are discussed for spin observables extracted from photoproduction of vector mesons. These constraints originate from positivity of the spin density matrix and should be part of any future analysis of experimental data. Spin observables need to be defined in the photon-nucleon c.m. frame.

  20. On Weighted Support Vector Regression

    DEFF Research Database (Denmark)

    Han, Xixuan; Clemmensen, Line Katrine Harder

    2014-01-01

    We propose a new type of weighted support vector regression (SVR), motivated by modeling local dependencies in time and space in prediction of house prices. The classic weights of the weighted SVR are added to the slack variables in the objective function (OF‐weights). This procedure directly...

  1. Portfolio Analysis for Vector Calculus

    Science.gov (United States)

    Kaplan, Samuel R.

    2015-01-01

    Classic stock portfolio analysis provides an applied context for Lagrange multipliers that undergraduate students appreciate. Although modern methods of portfolio analysis are beyond the scope of vector calculus, classic methods reinforce the utility of this material. This paper discusses how to introduce classic stock portfolio analysis in a…

  2. Large N Expansion. Vector Models

    OpenAIRE

    Nissimov, Emil; Pacheva, Svetlana

    2006-01-01

    Preliminary version of a contribution to the "Quantum Field Theory. Non-Perturbative QFT" topical area of "Modern Encyclopedia of Mathematical Physics" (SELECTA), eds. Aref'eva I, and Sternheimer D, Springer (2007). Consists of two parts - "main article" (Large N Expansion. Vector Models) and a "brief article" (BPHZL Renormalization).

  3. Vector-meson dominance revisited

    Directory of Open Access Journals (Sweden)

    Terschlüsen Carla

    2012-12-01

    Full Text Available The interaction of mesons with electromagnetism is often well described by the concept of vector-meson dominance (VMD. However, there are also examples where VMD fails. A simple chiral Lagrangian for pions, rho and omega mesons is presented which can account for the respective agreement and disagreement between VMD and phenomenology in the sector of light mesons.

  4. Sum rule analysis of vector and axial-vector spectral functions with excited states in vacuum

    OpenAIRE

    Hohler, Paul M.; Rapp, Ralf

    2012-01-01

    We simultaneously analyze vector and axial-vector spectral functions in vacuum using hadronic models constrained by experimental data and the requirement that Weinberg-type sum rules are satisfied. Upon explicit inclusion of an excited vector state, viz. rho', and the requirement that the perturbative continua are degenerate in vector and axial-vector channels, we deduce the existence of an excited axial-vector resonance state, a1', in order that the Weinberg sum rules are satisfied. The resu...

  5. Vector variational inequalities and their relations with vector optimization

    Directory of Open Access Journals (Sweden)

    Surjeet Kaur Suneja

    2014-01-01

    Full Text Available In this paper, K- quasiconvex, K- pseudoconvex and other related functions have been introduced in terms of their Clarke subdifferentials, where is an arbitrary closed convex, pointed cone with nonempty interior. The (strict, weakly -pseudomonotonicity, (strict K- naturally quasimonotonicity and K- quasimonotonicity of Clarke subdifferential maps have also been defined. Further, we introduce Minty weak (MVVIP and Stampacchia weak (SVVIP vector variational inequalities over arbitrary cones. Under regularity assumption, we have proved that a weak minimum solution of vector optimization problem (VOP is a solution of (SVVIP and under the condition of K- pseudoconvexity we have obtained the converse for MVVIP (SVVIP. In the end we study the interrelations between these with the help of strict K-naturally quasimonotonicity of Clarke subdifferential map.

  6. Introduction to vector velocity imaging

    DEFF Research Database (Denmark)

    Jensen, Jørgen Arendt; Udesen, Jesper; Hansen, Kristoffer Lindskov

    over the full region of interest and a real time image at a frame rate of 20 Hz can be displayed. Real time videos have been obtained from both our research systems and from commercial BK Medical scanners. The vector velocity images reveal the full complexity of the human blood flow. It is easy to see...... direction and the correct velocity magnitude for any orientation of the vessels. At complex geometries like bifurcations, branching and for valves the approach reveals how the velocity changes magnitude and direction over the cardiac cycle. Vector velocity reveals a wealth of new information that now...... is accessible to the ultrasound community. The displaying and studying of this information is challenging as complex flow changes rapidly over the cardiac cycle....

  7. Vector Galileon and inflationary magnetogenesis

    Science.gov (United States)

    Nandi, Debottam; Shankaranarayanan, S.

    2018-01-01

    Cosmological inflation provides the initial conditions for the structure formation. However, the origin of large-scale magnetic fields can not be addressed in this framework. The key issue for this long-standing problem is the conformal invariance of the electromagnetic (EM) field in 4-D. While many approaches have been proposed in the literature for breaking conformal invariance of the EM action, here, we provide a completely new way of looking at the modifications to the EM action and generation of primordial magnetic fields during inflation. We explicitly construct a higher derivative EM action that breaks conformal invariance by demanding three conditions—theory be described by vector potential Aμ and its derivatives, Gauge invariance be satisfied, and equations of motion be linear in second derivatives of vector potential. The unique feature of our model is that appreciable magnetic fields are generated at small wavelengths while tiny magnetic fields are generated at large wavelengths that are consistent with current observations.

  8. Clinical applications of power vectors.

    Science.gov (United States)

    Miller, Joseph M

    2009-06-01

    The study of infant vision is closely coupled to the study of the refraction, change in refraction over time, and the effect of spectacle correction on visual development. Frequently, reports are limited to descriptions of spherical equivalent or cylinder power without regard to axis, as data are frequently collected in the clinical format of sphere, cylinder, and axis (S, C, A). Conversion from clinical notation to a power vector representation of refraction allows unambiguous description of how refractions change over time and differ between repeated measurements. This article presents a series of examples of Microsoft Excel spreadsheet formulas that make the conversion from clinical notation to power vector format, and provides examples of useful applications of these methods.

  9. Active set support vector regression.

    Science.gov (United States)

    Musicant, David R; Feinberg, Alexander

    2004-03-01

    This paper presents active set support vector regression (ASVR), a new active set strategy to solve a straightforward reformulation of the standard support vector regression problem. This new algorithm is based on the successful ASVM algorithm for classification problems, and consists of solving a finite number of linear equations with a typically large dimensionality equal to the number of points to be approximated. However, by making use of the Sherman-Morrison-Woodbury formula, a much smaller matrix of the order of the original input space is inverted at each step. The algorithm requires no specialized quadratic or linear programming code, but merely a linear equation solver which is publicly available. ASVR is extremely fast, produces comparable generalization error to other popular algorithms, and is available on the web for download.

  10. Lentiviral vectors in cancer immunotherapy.

    Science.gov (United States)

    Oldham, Robyn Aa; Berinstein, Elliot M; Medin, Jeffrey A

    2015-01-01

    Basic science advances in cancer immunotherapy have resulted in various treatments that have recently shown success in the clinic. Many of these therapies require the insertion of genes into cells to directly kill them or to redirect the host's cells to induce potent immune responses. Other analogous therapies work by modifying effector cells for improved targeting and enhanced killing of tumor cells. Initial studies done using γ-retroviruses were promising, but safety concerns centered on the potential for insertional mutagenesis have highlighted the desire to develop other options for gene delivery. Lentiviral vectors (LVs) have been identified as potentially more effective and safer alternative delivery vehicles. LVs are now in use in clinical trials for many different types of inherited and acquired disorders, including cancer. This review will discuss current knowledge of LVs and the applications of this viral vector-based delivery vehicle to cancer immunotherapy.

  11. Vector fields on singular varieties

    CERN Document Server

    Brasselet, Jean-Paul; Suwa, Tatsuo

    2009-01-01

    Vector fields on manifolds play a major role in mathematics and other sciences. In particular, the Poincaré-Hopf index theorem gives rise to the theory of Chern classes, key manifold-invariants in geometry and topology. It is natural to ask what is the ‘good’ notion of the index of a vector field, and of Chern classes, if the underlying space becomes singular. The question has been explored by several authors resulting in various answers, starting with the pioneering work of M.-H. Schwartz and R. MacPherson. We present these notions in the framework of the obstruction theory and the Chern-Weil theory. The interplay between these two methods is one of the main features of the monograph.

  12. New Approach to Fractal Approximation of Vector-Functions

    National Research Council Canada - National Science Library

    Igudesman, Konstantin; Davletbaev, Marsel; Shabernev, Gleb

    2015-01-01

      This paper introduces new approach to approximation of continuous vector-functions and vector sequences by fractal interpolation vector-functions which are multidimensional generalization of fractal...

  13. Angles in Complex Vector Spaces

    OpenAIRE

    Scharnhorst, K.

    1999-01-01

    The article reviews some of the (fairly scattered) information available in the mathematical literature on the subject of angles in complex vector spaces. The following angles and their relations are considered: Euclidean, complex, and Hermitian angles, (Kasner's) pseudo-angle, the Kaehler angle (synonyms for the latter used in the literature are: angle of inclination, characteristic deviation, holomorphic deviation, holomorphy angle, Wirtinger angle, slant angle).

  14. GAPS IN SUPPORT VECTOR OPTIMIZATION

    Energy Technology Data Exchange (ETDEWEB)

    STEINWART, INGO [Los Alamos National Laboratory; HUSH, DON [Los Alamos National Laboratory; SCOVEL, CLINT [Los Alamos National Laboratory; LIST, NICOLAS [Los Alamos National Laboratory

    2007-01-29

    We show that the stopping criteria used in many support vector machine (SVM) algorithms working on the dual can be interpreted as primal optimality bounds which in turn are known to be important for the statistical analysis of SVMs. To this end we revisit the duality theory underlying the derivation of the dual and show that in many interesting cases primal optimality bounds are the same as known dual optimality bounds.

  15. Ultrasonic Dynamic Vector Stress Sensor

    Science.gov (United States)

    Heyman, Joseph S.; Froggatt, Mark

    1992-01-01

    Stress inferred from measurements in specimens rather than in bonded gauges. Ultrasonic dynamic vector stress sensor (UDVSS) measures changes in dynamic directional stress occurring in material or structure at location touched by device when material or structure put under cyclic load. Includes phase-locked loop, synchronous amplifier, and contact probe. Useful among manufacturers of aerospace and automotive structures for stress testing and evaluation of designs.

  16. A ocean bottom vector magnetometer

    Science.gov (United States)

    Wang, Xiaomei; Teng, Yuntian; Wang, Chen; Ma, Jiemei

    2017-04-01

    The new development instrument with a compact spherical coil system and Overhauser magnetometer for measuring the total strength of the magnetic field and the vectors of strength, Delta inclination - Delta declination, meanwhile we also use a triaxial fluxgate instrument of the traditional instrument for geomagnetic vector filed measurement. The advantages of this method are be calibrated by each other and get good performances with automatic operation, good stability and high resolution. Firstly, a brief description of the instrument measurement principles and the key technologies are given. The instrument used a spherical coil system with 34 coils to product the homogeneous volume inside the coils which is large enough to accommodate the sensor of Overhauser total field sensor; the rest of the footlocker-sized ocean-bottom vector magnetometer consists of equipment to run the sensors and records its data (batteries and a data logger), weight to sink it to the sea floor, a remote-controlled acoustic release and flotation to bring the instrument back to the surface. Finally, the accuracy of the instrument was tested in the Geomagnetic station, and the measurement accuracies of total strength and components were better than 0.2nT and 1nT respectively. The figure 1 shows the development instrument structure. it includes six thick glass spheres which protect the sensor, data logger and batteries from the pressures of the deep sea, meanwhile they also provide recycling positive buoyancy; To cushion the glass, the spheres then go inside yellow plastic "hardhats". The triaxial fluxgate is inside No.1 glass spheres, data logger and batteries are inside No.2 glass spheres, the new vector sensor is inside No.3 glass spheres, acoustic communication unit is inside No.4 glass spheres, No.5 and No.6 glass spheres are empty which only provide recycling positive buoyancy. The figure 2 shows the development instrument Physical photo.

  17. Olfaction in vector-host interactions

    NARCIS (Netherlands)

    Takken, W.; Knols, B.G.J.

    2010-01-01

    This book addresses the topic how blood-feeding arthropods interact with their vertebrate hosts. As the transmission of infectious vector-borne pathogens is much dependent on the contact between vector and host, the efficacy of host location is of profound importance. Interruption of vector-host

  18. Herbivore arthropods benefit from vectoring plant viruses

    NARCIS (Netherlands)

    Belliure, B.; Janssen, A.; Maris, P.C.; Peters, D.; Sabelis, M.W.

    2005-01-01

    Plants infected with pathogens often attract the pathogens' vectors, but it is not clear if this is advantageous to the vectors. We therefore quantified the direct and indirect (through the host plant) effects of a pathogen on its vector. A positive direct effect of the plant-pathogenic Tomato

  19. Vector and tensor analysis with applications

    CERN Document Server

    Borisenko, A I; Silverman, Richard A

    1979-01-01

    Concise and readable, this text ranges from definition of vectors and discussion of algebraic operations on vectors to the concept of tensor and algebraic operations on tensors. It also includes a systematic study of the differential and integral calculus of vector and tensor functions of space and time. Worked-out problems and solutions. 1968 edition.

  20. New recombinant serotypes of AAV vectors.

    Science.gov (United States)

    Gao, Guangping; Vandenberghe, Luk H; Wilson, James M

    2005-06-01

    AAV based vectors can achieve stable gene transfer with minimal vector related toxicities. AAV serotype 2 (AAV2) is the first AAV that was vectored for gene transfer applications. However, the restricted tissue tropism of AAV and its low transduction efficiency have limited its further development as vector. Recent studies using vectors derived from alternative AAV serotypes such as AAV1, 4, 5 and 6 have shown improved potency and broadened tropism of the AAV vector by packaging the same vector genome with different AAV capsids. In an attempt to search for potent AAV vectors with enhanced performance profiles, molecular techniques were employed for the detection and isolation of endogenous AAVs from a variety of human and non-human primate (NHP) tissues. A family of novel primate AAVs consisting of 110 non-redundant species of proviral sequences was discovered and turned to be prevalent in 18-19% of the tissues evaluated. Phylogenetic and functional analyses revealed that primate AAVs are segregated into clades based on phylogenetic relatedness. The members within a clade share functional and serological properties. Initial evaluation in mouse models of vectors based on these novel AAVs for tissue tropism and gene transfer potency led to the identification of some vector with improved gene transfer to different target tissues. Gene therapy treatment of several mouse and canine models with novel AAV vectors achieved long term phenotypic corrections. Vectors based on new primate AAVs could become the next generation of efficient gene transfer vehicles for various gene therapy applications.

  1. Vectorizing and machine-spanning techniques

    Energy Technology Data Exchange (ETDEWEB)

    Giroux, E D

    1983-09-22

    Techniques for vectorizing complex logic are shown using a decoupled sliding-surface calculation that is part of a two-dimensional Lagrangian simulation model. The same source coding can be run on many vector, parallel, and multiprocessor computers with very little or no alteration. The vectorizing techniques have been used for a wide range of problems.

  2. Fubini's Theorem for Vector-Valued Measures

    Science.gov (United States)

    Uglanov, A. V.

    1991-02-01

    The situation is considered when either the transitional or initial measure is vector-valued (the other is, respectively, scalar-valued; thus the product measure is also vector-valued). The integrable function is vector-valued. In this situation two theorems of Fubini type are proved.

  3. The changing dielectric properties of CHO cells can be used to determine early apoptotic events in a bioprocess.

    Science.gov (United States)

    Braasch, Katrin; Nikolic-Jaric, Marija; Cabel, Tim; Salimi, Elham; Bridges, Greg E; Thomson, Doug J; Butler, Michael

    2013-11-01

    To ensure maximum productivity of recombinant proteins it is desirable to prolong cell viability during a mammalian cell bioprocess, and therefore important to carefully monitor cell density and viability. In this study, five different and independent methods of monitoring were applied to Chinese hamster ovary (CHO) cells grown in a batch culture in a controlled bioreactor to determine cell density and/or cell viability. They included: a particle counter, trypan blue exclusion (Cedex), an in situ bulk capacitance probe, an off-line fluorescent flow cytometer, and a prototype dielectrophoretic (DEP) cytometer. These various techniques gave similar values during the exponential growth phase. However, beyond the exponential growth phase the viability measurements diverged. Fluorescent flow cytometry with a range of fluorescent markers was used to investigate this divergence and to establish the progress of cell apoptosis: the cell density estimates by the intermediate stage apoptosis assay agreed with those obtained by the bulk capacitance probe and the early stage apoptosis assay viability measurements correlated well with the DEP cytometer. The trypan blue assay showed higher estimates of viable cell density and viability compared to the capacitance probe or the DEP cytometer. The DEP cytometer measures the dielectric properties of individual cells and identified at least two populations of cells, each with a distinct polarizability. As verified by comparison with the Nexin assay, one population was associated with viable (non-apoptotic) cells and the other with apoptotic cells. From the end of the exponential through the stationary and decline stages there was a gradual shift of cell count from the viable into the apoptotic population. However, the two populations maintained their individual dielectric properties throughout this shift. This leads to the conclusion that changes in bulk dielectric properties of cultures might be better modeled as shifts in cells

  4. Comparative study of the cytotoxic and genotoxic effects of titanium oxide and aluminium oxide nanoparticles in Chinese hamster ovary (CHO-K1) cells

    Energy Technology Data Exchange (ETDEWEB)

    Di Virgilio, A.L. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA), Diag. 113 y 64, Correo 16, Suc. 4, La Plata (1900) (Argentina); Reigosa, M. [Instituto Multidisciplinario de Biologia Celular (IMBICE), Calle 526 y Camino Gral. Belgrano (entre 10 y 11), La Plata 1900 (Argentina); Arnal, P.M. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA), Diag. 113 y 64, Correo 16, Suc. 4, La Plata 1900 (Argentina); Fernandez Lorenzo de Mele, M., E-mail: mmele@inifta.unlp.edu.ar [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA), Diag. 113 y 64, Correo 16, Suc. 4, La Plata 1900 (Argentina)

    2010-05-15

    The aim of this study was to analyze the cytotoxicity and genotoxicity of titanium oxide (TiO{sub 2}) and aluminium oxide (Al{sub 2}O{sub 3}) nanoparticles (NPs) on Chinese hamster ovary (CHO-K1) cells using neutral red (NR), mitochondrial activity (by MTT assay), sister chromatid exchange (SCE), micronucleus (MN) formation, and cell cycle kinetics techniques. Results showed a dose-related cytotoxic effect evidenced after 24 h by changes in lysosomal and mitochondrial dehydrogenase activity. Interestingly, transmission electronic microscopy (TEM) showed the formation of perinuclear vesicles in CHO-K1 cells after treatment with both NPs during 24 h but no NP was detected in the nuclei. Genotoxic effects were shown by MN frequencies which significantly increased at 0.5 and 1 {mu}g/mL TiO{sub 2} and 0.5-10 {mu}g/mL Al{sub 2}O{sub 3}. SCE frequencies were higher for cells treated with 1-5 {mu}g/mL TiO{sub 2}. The absence of metaphases evidenced cytotoxicity for higher concentrations of TiO{sub 2}. No SCE induction was achieved after treatment with 1-25 {mu}g/mL Al{sub 2}O{sub 3}. In conclusion, findings showed cytotoxic and genotoxic effects of TiO{sub 2} and Al{sub 2}O{sub 3} NPs on CHO-K1 cells. Possible causes of controversial reports are discussed further on.

  5. Advancement in recombinant protein production using a marine oxygen carrier to enhance oxygen transfer in a CHO-S cell line.

    Science.gov (United States)

    Le Pape, Fiona; Bossard, Morgane; Dutheil, Delphine; Rousselot, Morgane; Polard, Valérie; Férec, Claude; Leize, Elisabeth; Delépine, Pascal; Zal, Franck

    2015-06-01

    Recombinant proteins, particularly proteins used as therapeutics, are widely expressed for bioprocessing manufacturing processes. Mammalian cell lines represent the major host cells for bioproduction, according to their capacities of post-translational modifications and folding of secreted proteins. Many parameters can affect cell productivity, especially the rate of oxygen transfer. Dissolved oxygen, in high or low proportions, is a crucial parameter which can affect cell viability and thus productivity. HEMARINA has developed a new technology, commercially proposed as HEMOXCell(®), to improve cell culture at a large production scale. HEMOXCell(®) is a marine oxygen carrier having properties of high oxygen sensitivity, to be used as an oxygen additive during cell culture manufacturing. In this study, we investigated the effects of HEMOXCell(®) on the culture of the commonly used CHO-S cell line. Two main objectives were pursued: 1) cell growth rate and viability during a batch mode process, and 2) the determination of the effect of this oxygen carrier on recombinant protein production from a CHO-transfected cell line. Our results show an increase of CHO-S cellular growth at a rate of more than four-fold in culture with HEMOXCell(®). Moreover, an extension of the growth exponential phase and high cell viability were observed. All of these benefits seem to contribute to the improvement of recombinant protein production. This work underlines several applications using this marine-type oxygen carrier for large biomanufacturing. It is a promising cell culture additive according to the increasing demand for therapeutic products such as monoclonal antibodies.

  6. Macrophage receptor with collagenous structure (MARCO) is a dynamic adhesive molecule that enhances uptake of carbon nanotubes by CHO-K1 Cells

    Energy Technology Data Exchange (ETDEWEB)

    Hirano, Seishiro, E-mail: seishiro@nies.go.jp [Environmental Nanotoxicology Project, RCER, National Institute for Environmental Studies (Japan); Fujitani, Yuji; Furuyama, Akiko [Environmental Nanotoxicology Project, RCER, National Institute for Environmental Studies (Japan); Kanno, Sanae [Department of Legal Medicine, St. Marianna School of Medicine (Japan)

    2012-02-15

    The toxicity of carbon nanotubes (CNTs), a highly promising nanomaterial, is similar to that of asbestos because both types of particles have a fibrous shape and are biopersistent. Here, we investigated the characteristics of macrophage receptor with collagenous structure (MARCO), a membrane receptor expressed on macrophages that recognizes environmental or unopsonized particles, and we assessed whether and how MARCO was involved in cellular uptake of multi-walled CNTs (MWCNTs). MARCO-transfected Chinese hamster ovary (CHO-K1) cells took up polystyrene beads irrespective of the particle size (20 nm–1 μm). In the culture of MARCO-transfected CHO-K1 cells dendritic structures were observed on the bottom of culture dishes, and the edges of these dendritic structures were continually renewed as the cell body migrated along the dendritic structures. MWCNTs were first tethered to the dendritic structures and then taken up by the cell body. MWCNTs appeared to be taken up via membrane ruffling like macropinocytosis, rather than phagocytosis. The cytotoxic EC{sub 50} value of MWCNTs in MARCO-transfected CHO-K1 cells was calculated to be 6.1 μg/mL and transmission electron microscopic observation indicated that the toxicity of MWCNTs may be due to the incomplete inclusion of MWCNTs by the membrane structure. -- Highlights: ►Carbon nanotubes (CNTs) were tethered to MARCO in vitro. ►CNTs were taken up rapidly into the cell body via MARCO by membrane ruffling. ►The incomplete inclusion of CNTs by membranes caused cytotoxicity.

  7. miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

    Science.gov (United States)

    Fischer, Simon; Marquart, Kim F; Pieper, Lisa A; Fieder, Juergen; Gamer, Martin; Gorr, Ingo; Schulz, Patrick; Bradl, Harald

    2017-07-01

    In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495-1510. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  8. Quantitative mammalian cell mutagenesis and a preliminary study of the mutagenic potential of metallic compounds. [Cell system used was CHO/HGPRT

    Energy Technology Data Exchange (ETDEWEB)

    Hsie, A.W.; Johnson, N.P.; Couch, D.B.; San Sebastian, J.R.; O' Neill, J.P.; Forbes, N.L.

    1978-01-01

    We have defined a set of stringent conditions required to quantify specific gene mutation in a mammalian cell system, CHO/HGPRT. Greater than 98% of the 6-thioguanine (TG)-resistant variants were shown to be deficient in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in Chinese hamster ovary (CHO) cells. The sensitive and quantitative nature of this assay was utilized to study the structure-activity (mutagenicity) relationship of various classes of chemicals. Mutagenicity as determined in the CHO/HGPRT assay, appears to correlate well (76/83 (92%)) with the reported animal carcinogenicity of 108 chemicals studied. The system also appears to be suitable for studying the mutagenicity and cytotoxicity of metallic compounds. We found that cis-dichlorodiammine Pt(II) (cis-Pt(NH/sub 3/)/sub 2/Cl/sub 2/) (cis-DDP), one of the widely used inorganic antitumor agents, is cytotoxic and mutagenic. Mutagenicity of cis-DDP correlates with its binding to DNA. However, trans-DDP, (Pt(NH/sub 3/)/sub 4/)Cl/sub 2/, and K/sub 2/(PtCl/sub 4/) exhibit greatly reduced biological activities. Among 14 other metals studied, we found that carcinogenic metallic compounds, such as MnCl/sub 2/, NiCl/sub 2/, and BeSO/sub 4/ are mutagenic, while non-carcinogenic compounds such as MgCl/sub 2/ and H/sub 2/SeO/sub 3/ are not. Determination of metal mutagenicity is apparently complicated by the ionic composition of the medium. This may account in part for varying results in studies of the mutagenicity of other metallic compounds. Further refinement of the assay conditions, especially with respect to the ionic environment necessary for quantifying mutagenesis of each metallic agent, is in progress.

  9. Anti-cell death engineering of CHO cells: co-overexpression of Bcl-2 for apoptosis inhibition, Beclin-1 for autophagy induction.

    Science.gov (United States)

    Lee, Jae Seong; Ha, Tae Kwang; Park, Jin Hyoung; Lee, Gyun Min

    2013-08-01

    Genetic engineering approaches to inhibit cell death in Chinese hamster ovary (CHO) cell cultures have been limited primarily to anti-apoptosis engineering. Recently, autophagy has received attention as a new anti-cell death engineering target in addition to apoptosis. In order to achieve a more efficient protection of cells from the stressful culture conditions, the simultaneous targeting of anti-apoptosis and pro-autophagy in CHO cells (DG44) was attempted by co-overexpressing an anti-apoptotic protein, Bcl-2, and a key regulator of autophagy pathway, Beclin-1, respectively. Co-overexpression of Bcl-2 and Beclin-1 exhibited a longer culture period as well as higher viability during serum-free suspension culture, compared with the control (without co-overexpression of Bcl-2 and Beclin-1) and Bcl-2 overexpression only. In addition to the efficient inhibition of apoptosis by Bcl-2 overexpression, Beclin-1 overexpression successfully induced the increase in the autophagic marker protein, LC3-II, and autophagosome formation with the decrease in mTOR activity. Co-immunoprecipitation and qRT-PCR experiments revealed that the enforced expression of Beclin-1 increased Ulk1 expression and level of free-Beclin-1 that did not bind to the Bcl-2 despite the Bcl-2 overexpression. Under other stressful culture conditions such as treatment with sodium butyrate and hyperosmolality, co-overexpression of Bcl-2 and Beclin-1 also protected the cells from cell death more efficiently than Bcl-2 overexpression only, implying the potential of autophagy induction. Taken together, the data obtained here provide the evidence that pro-autophagy engineering together with anti-apoptosis engineering yields a synergistic effect and successfully enhances the anti-cell death engineering of CHO cells. Copyright © 2013 Wiley Periodicals, Inc.

  10. On the Distribution of Norm of Vector Projection and Rejection of Two Complex Normal Random Vectors

    Directory of Open Access Journals (Sweden)

    Mehdi Maleki

    2015-01-01

    Full Text Available Vector projection and vector rejection are highly common and useful operations in mathematics, information theory, and signal processing. In this paper, we find the distribution of the norm of projection and rejection vectors when the original vectors are standard complex normally distributed.

  11. Gaussian statistics for palaeomagnetic vectors

    Science.gov (United States)

    Love, J.J.; Constable, C.G.

    2003-01-01

    With the aim of treating the statistics of palaeomagnetic directions and intensities jointly and consistently, we represent the mean and the variance of palaeomagnetic vectors, at a particular site and of a particular polarity, by a probability density function in a Cartesian three-space of orthogonal magnetic-field components consisting of a single (unimoda) non-zero mean, spherically-symmetrical (isotropic) Gaussian function. For palaeomagnetic data of mixed polarities, we consider a bimodal distribution consisting of a pair of such symmetrical Gaussian functions, with equal, but opposite, means and equal variances. For both the Gaussian and bi-Gaussian distributions, and in the spherical three-space of intensity, inclination, and declination, we obtain analytical expressions for the marginal density functions, the cumulative distributions, and the expected values and variances for each spherical coordinate (including the angle with respect to the axis of symmetry of the distributions). The mathematical expressions for the intensity and off-axis angle are closed-form and especially manageable, with the intensity distribution being Rayleigh-Rician. In the limit of small relative vectorial dispersion, the Gaussian (bi-Gaussian) directional distribution approaches a Fisher (Bingham) distribution and the intensity distribution approaches a normal distribution. In the opposite limit of large relative vectorial dispersion, the directional distributions approach a spherically-uniform distribution and the intensity distribution approaches a Maxwell distribution. We quantify biases in estimating the properties of the vector field resulting from the use of simple arithmetic averages, such as estimates of the intensity or the inclination of the mean vector, or the variances of these quantities. With the statistical framework developed here and using the maximum-likelihood method, which gives unbiased estimates in the limit of large data numbers, we demonstrate how to

  12. Comparison of five different in vitro assays for assessment of sodium metavanadate cytotoxicity in Chinese hamster ovary cells (CHO-K1 line).

    Science.gov (United States)

    Zwolak, Iwona

    2015-08-01

    This investigation was undertaken to compare five different in vitro cytotoxicity assays for their power in revealing vanadium-mediated toxicity in Chinese hamster ovary (CHO)-K1 cells. The cells were exposed to sodium metavanadate (NaVO(3)) in the range of 10-1000 µM for 24 h and thereafter the cytotoxic effects of NaVO(3) were measured by colorimetric in vitro assays: the neutral red (NR) test, the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) assay, the resazurin assay, the sulforhodamine B (SR-B) assay, and by microscopic assessment of cell viability using the trypan blue (TB) staining method. Among the assays used, the NR test was the most sensitive, since it revealed metavanadate cytotoxicity at the lowest NaVO(3) dose (=50 µM). Also, NaVO(3) cytotoxicity expressed as inhibitory concentration (IC) showed the lowest values for the NR test. Three other tests XTT, resazurin, and SR-B assays showed intermediate sensitivity revealing the cytotoxicity of NaVO(3) at 100 µM. The corresponding IC10 and IC50 values calculated for the XTT, resazurin, and SR-B tests were similar. The TB staining method was the least sensitive, since it recorded metavanadate cytotoxicity at the highest NaVO(3) concentration tested (=600 µM). Based on the cytotoxicity end points measured with the above assays, it can be concluded that lysosomal/Golgi apparatus damage (measured by NR assay) may be the primary effect of NaVO(3) on CHO-K1 cells. The disintegration of mitochondria (assessed with the XTT and resazurin assays) probably follows lysosomal impairment. Plasma membrane permeability (staining with TB) occurs at a late stage of NaVO(3)-induced cytotoxicity on CHO-K1 cells. The results obtained in this research work show that the NR test can be recommended as a very sensitive assay for the assessment of NaVO(3) cytotoxicity in the CHO-K1 cell culture model. Considering the convenience of assay performance along with adequate sensitivity

  13. Problems and worked solutions in vector analysis

    CERN Document Server

    Shorter, LR

    2014-01-01

    ""A handy book like this,"" noted The Mathematical Gazette, ""will fill a great want."" Devoted to fully worked out examples, this unique text constitutes a self-contained introductory course in vector analysis for undergraduate and graduate students of applied mathematics.Opening chapters define vector addition and subtraction, show how to resolve and determine the direction of two or more vectors, and explain systems of coordinates, vector equations of a plane and straight line, relative velocity and acceleration, and infinitely small vectors. The following chapters deal with scalar and vect

  14. Integration of vector-valued functions with respect to vector measures defined on δ-rings

    OpenAIRE

    Chakraborty, N. D.; Basu, Santwana

    2011-01-01

    This paper extends the theory of scalar-valued integrable functions with respect to vector measures defined on δ-rings to the case of vector-valued tensor integrable functions with respect to vector measures defined on δ-rings. This paper also generalizes some results of G. F. Stefánsson for tensor integration theory of vector-valued functions with respect to vector measures defined on σ-algebras.

  15. Multiscale vector fields for image pattern recognition

    Science.gov (United States)

    Low, Kah-Chan; Coggins, James M.

    1990-01-01

    A uniform processing framework for low-level vision computing in which a bank of spatial filters maps the image intensity structure at each pixel into an abstract feature space is proposed. Some properties of the filters and the feature space are described. Local orientation is measured by a vector sum in the feature space as follows: each filter's preferred orientation along with the strength of the filter's output determine the orientation and the length of a vector in the feature space; the vectors for all filters are summed to yield a resultant vector for a particular pixel and scale. The orientation of the resultant vector indicates the local orientation, and the magnitude of the vector indicates the strength of the local orientation preference. Limitations of the vector sum method are discussed. Investigations show that the processing framework provides a useful, redundant representation of image structure across orientation and scale.

  16. Rotations with Rodrigues' vector

    Energy Technology Data Exchange (ETDEWEB)

    Pina, E, E-mail: pge@xanum.uam.mx [Prof. Eugenio Mendez Docurro de la, Escuela Superior de Fisica y Matematicas del IPN, Zacatenco 07738, Mexico DF (Mexico)

    2011-09-15

    The rotational dynamics was studied from the point of view of Rodrigues' vector. This vector is defined here by its connection with other forms of parametrization of the rotation matrix. The rotation matrix was expressed in terms of this vector. The angular velocity was computed using the components of Rodrigues' vector as coordinates. It appears to be a fundamental matrix that is used to express the components of the angular velocity, the rotation matrix and the angular momentum vector. The Hamiltonian formalism of rotational dynamics in terms of this vector uses the same matrix. The quantization of the rotational dynamics is performed with simple rules if one uses Rodrigues' vector and similar formal expressions for the quantum operators that mimic the Hamiltonian classical dynamics.

  17. Transversals of Complex Polynomial Vector Fields

    DEFF Research Database (Denmark)

    Dias, Kealey

    Vector fields in the complex plane are defined by assigning the vector determined by the value P(z) to each point z in the complex plane, where P is a polynomial of one complex variable. We consider special families of so-called rotated vector fields that are determined by a polynomial multiplied...... by rotational constants. Transversals are a certain class of curves for such a family of vector fields that represent the bifurcation states for this family of vector fields. More specifically, transversals are curves that coincide with a homoclinic separatrix for some rotation of the vector field. Given...... examples of rotated families to argue this. There will be discussed several open questions concerning the number of transversals that can appear for a certain degree d of a polynomial vector field, and furthermore how transversals are analyzed with respect to bifurcations around multiple equilibrium points....

  18. A novel system for simultaneous or sequential integration of multiple gene-loading vectors into a defined site of a human artificial chromosome.

    Directory of Open Access Journals (Sweden)

    Teruhiko Suzuki

    Full Text Available Human artificial chromosomes (HACs are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming.

  19. Deciphering Babesia-Vector Interactions

    Directory of Open Access Journals (Sweden)

    Sandra Antunes

    2017-09-01

    Full Text Available Understanding host-pathogen-tick interactions remains a vitally important issue that might be better understood by basic research focused on each of the dyad interplays. Pathogens gain access to either the vector or host during tick feeding when ticks are confronted with strong hemostatic, inflammatory and immune responses. A prominent example of this is the Babesia spp.—tick—vertebrate host relationship. Babesia spp. are intraerythrocytic apicomplexan organisms spread worldwide, with a complex life cycle. The presence of transovarial transmission in almost all the Babesia species is the main difference between their life cycle and that of other piroplasmida. With more than 100 species described so far, Babesia are the second most commonly found blood parasite of mammals after trypanosomes. The prevalence of Babesia spp. infection is increasing worldwide and is currently classified as an emerging zoonosis. Babesia microti and Babesia divergens are the most frequent etiological agents associated with human babesiosis in North America and Europe, respectively. Although the Babesia-tick system has been extensively researched, the currently available prophylactic and control methods are not efficient, and chemotherapeutic treatment is limited. Studying the molecular changes induced by the presence of Babesia in the vector will not only elucidate the strategies used by the protozoa to overcome mechanical and immune barriers, but will also contribute toward the discovery of important tick molecules that have a role in vector capacity. This review provides an overview of the identified molecules involved in Babesia-tick interactions, with an emphasis on the fundamentally important ones for pathogen acquisition and transmission.

  20. Bridging the Vector Calculus Gap

    Science.gov (United States)

    Dray, Tevian; Manogue, Corinne

    2003-05-01

    As with Britain and America, mathematicians and physicists are separated from each other by a common language. In a nutshell, mathematics is about functions, but physics is about things. For the last several years, we have led an NSF-supported effort to "bridge the vector calculus gap" between mathematics and physics. The unifying theme we have discovered is to emphasize geometric reasoning, not (just) algebraic computation. In this talk, we will illustrate the language differences between mathematicians and physicists, and how we are trying reconcile them in the classroom. For further information about the project go to: http://www.physics.orst.edu/bridge

  1. Probing the importance of clonality: Single cell subcloning of clonally derived CHO cell lines yields widely diverse clones differing in growth, productivity, and product quality.

    Science.gov (United States)

    Ko, Peggy; Misaghi, Shahram; Hu, Zhilan; Zhan, Dejin; Tsukuda, Joni; Yim, Mandy; Sanford, Mark; Shaw, David; Shiratori, Masaru; Snedecor, Brad; Laird, Michael; Shen, Amy

    2017-12-11

    In the past few decades, a large variety of therapeutic antibodies and proteins have been expressed in Chinese hamster ovary (CHO) cells. This mammalian expression system is robust, scalable, relatively inexpensive, and importantly allows for post-translational modifications that are important for some therapeutic proteins. Historically, CHO cell lines were derived from colonies of cells grown in semi-solid or liquid plates using either serum-containing or serum-free media. Current advancements in cell sorting and imaging technologies have allowed for isolating and imaging single cell progenitors at the seeding step, significantly increasing the probability of isolating clonally derived cell lines. However, it is debatable how much population heterogeneity can be eliminated when clonally derived cell lines, originated from a single cell progenitor, are scaled up. To further investigate this phenomenon, we subcloned two different clonally derived (day 0 imaged and visually inspected) cell lines expressing antibody-X. The results showed that when six randomly chosen subclones of each line were evaluated in a production assay, these subclones displayed a range of variation in titer, specific productivity, growth, and product quality attributes. Some subclones displayed variations in transgene copy numbers. Additionally, clonal derivation did not assure stability of the derived cell lines. Our findings show that cell heterogeneity exists in a population even when derived from a single cell progenitor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

  2. DNA display selection of peptide ligands for a full-length human G protein-coupled receptor on CHO-K1 cells.

    Directory of Open Access Journals (Sweden)

    Nobuhide Doi

    Full Text Available The G protein-coupled receptors (GPCRs, which form the largest group of transmembrane proteins involved in signal transduction, are major targets of currently available drugs. Thus, the search for cognate and surrogate peptide ligands for GPCRs is of both basic and therapeutic interest. Here we describe the application of an in vitro DNA display technology to screening libraries of peptide ligands for full-length GPCRs expressed on whole cells. We used human angiotensin II (Ang II type-1 receptor (hAT1R as a model GPCR. Under improved selection conditions using hAT1R-expressing Chinese hamster ovary (CHO-K1 cells as bait, we confirmed that Ang II gene could be enriched more than 10,000-fold after four rounds of selection. Further, we successfully selected diverse Ang II-like peptides from randomized peptide libraries. The results provide more precise information on the sequence-function relationships of hAT1R ligands than can be obtained by conventional alanine-scanning mutagenesis. Completely in vitro DNA display can overcome the limitations of current display technologies and is expected to prove widely useful for screening diverse libraries of mutant peptide and protein ligands for receptors that can be expressed functionally on the surface of CHO-K1 cells.

  3. Sustained productivity in recombinant Chinese Hamster Ovary (CHO cell lines: proteome analysis of the molecular basis for a process-related phenotype

    Directory of Open Access Journals (Sweden)

    Gammell Patrick

    2011-07-01

    Full Text Available Abstract Background The ability of mammalian cell lines to sustain cell specific productivity (Qp over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture. Results Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis and LC-MS/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3 that were differentially expressed in the same direction. Conclusion These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.

  4. Effects of Peptone Supplementation in Different Culture Media on Growth, Metabolic Pathway and Productivity of CHO DG44 Cells; a New Insight into Amino Acid Profiles.

    Science.gov (United States)

    Davami, Fatemeh; Eghbalpour, Farnaz; Nematollahi, Leila; Barkhordari, Farzaneh; Mahboudi, Fereidoun

    2015-01-01

    The optimization of bioprocess conditions towards improved growth profile and productivity yield is considered of great importance in biopharmaceutical manufacturing. Peptones as efficient sources of nutrients have been studied for their effect on media development; however, their role on metabolic pathway is not well understood. In the present study, the effect of different concentration of peptones on a recombinant Chinese hamster ovary (CHO) cell line grown in three serum-free suspension cultures was determined. Six peptones of different origins and available amino acid profiles were investigated regarding their impact on cell growth, productivity, and metabolic pathways changes. In optimized feeding strategies, increases of 136% and 159% in volumetric productivity (for a low-nutrient culture media) and 55% (for a high-nutrient culture media) were achieved. Furthermore, particular sources of peptones with specific amino acid profile developed preferential results for each different culture medium. Two peptones, SoyA2SC and SoyE-110, were the only hydrolysates that showed production improvement in all three media. Casein Peptone plus Tryptone N1 and SoyA3SC showed different improved results based on their implemented concentration for each individual basal medium. The amino acid profile of peptones may provide clues to identify the most effective feeding strategies for recombinant CHO cells.

  5. Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives.

    Directory of Open Access Journals (Sweden)

    Lena Thoring

    Full Text Available Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called "difficult-to-express" proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of "difficult-to-express" proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called "cell-free" protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various "difficult-to-express" proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA.

  6. Sustained productivity in recombinant Chinese Hamster Ovary (CHO) cell lines: proteome analysis of the molecular basis for a process-related phenotype

    LENUS (Irish Health Repository)

    Meleady, Paula

    2011-07-24

    Abstract Background The ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture. Results Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS\\/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction. Conclusion These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.

  7. Preselection of recombinant gene integration sites enabling high transcription rates in CHO cells using alternate start codons and recombinase mediated cassette exchange.

    Science.gov (United States)

    Baumann, Martina; Gludovacz, Elisabeth; Sealover, Natalie; Bahr, Scott; George, Henry; Lin, Nan; Kayser, Kevin; Borth, Nicole

    2017-11-01

    Site-specific recombinase mediated cassette exchange (RMCE) enables the transfer of the gene of interest (GOI) into pre-selected genomic locations with defined expression properties. For the generation of recombinant production cell lines, this has the advantage that screening for high transcription rates at the genome integration site would be required only once, with the possibility to reuse the selected site for new products. Here, we describe a strategy that aims at the selection of transcriptionally active genome integration sites in Chinese Hamster Ovary (CHO) cells by using alternate start codons in the surface reporter protein CD4, in combination with FACS sorting for high expressers. The alternate start codon reduces the translation initiation efficiency and allows sorting for CHO cells with the highest transcription rates, while RMCE enables the subsequent exchange of the CD4 against the GOI. We have shown that sorted cell pools with the CD4 reporter gene containing the alternate start codon CTG lead to higher GFP signals and higher antibody titers upon RMCE as compared to cell pools containing the ATG start codon of the CD4 reporter. Despite the absence of any subcloning step, the final cell pool contained the CD4 gene in a single genome integration site. © 2017 Wiley Periodicals, Inc.

  8. Study of potential inhibitors of thyroid iodide uptake by using CHO cells stably expressing the human sodium/iodide symporter (hNIS) protein.

    Science.gov (United States)

    Agretti, P; Dimida, A; De Marco, G; Ferrarini, E; Rodrìguez Gonzàlez, J C; Santini, F; Vitti, P; Pinchera, A; Tonacchera, M

    2011-03-01

    Thyroid gland is highly dependent on dietary intake of iodine for normal function, so it is particularly subjected to "endocrine disruptor" action. The human sodium/iodide symporter (hNIS) is an integral plasma membrane glycoprotein mediating the active transport of iodide into thyroid follicular cells, a crucial step for thyroid hormone biosynthesis. Beyond to perchlorate and thyocianate ions a few other inhibitors of iodide uptake have been described. The aim of this study was to investigate if 10 substances usually used as drugs in clinical practice were able to inhibit NIS-mediated iodide uptake in vitro. A CHO cell line stably expressing hNIS was used to test any inhibition of NIS-mediated iodide uptake exerted by drugs. Perchlorate and thyocianate ions were used as positive controls. None of the analyzed substances was able to significantly inhibit iodide uptake in our system. As we expected, perchlorate and thyocianate ions were able to inhibit iodide uptake in a dose-dependent manner. In conclusion, we carried out an in vitro assay to evaluate the potential inhibitory effect of common drugs on NISmediated iodide uptake by using CHO-hNIS cells. None of the analyzed substances was able to inhibit iodide uptake; only perchlorate and thyocianate were able to inhibit iodide uptake in a dose-dependent manner.

  9. Communication: Photodissociation of CH3CHO at 308 nm: Observation of H-roaming, CH3-roaming, and transition state pathways together along the ground state surface

    Science.gov (United States)

    Li, Hou-Kuan; Tsai, Po-Yu; Hung, Kai-Chan; Kasai, Toshio; Lin, King-Chuen

    2015-01-01

    Following photodissociation of acetaldehyde (CH3CHO) at 308 nm, the CO(v = 1-4) fragment is acquired using time-resolved Fourier-transform infrared emission spectroscopy. The CO(v = 1) rotational distribution shows a bimodal feature; the low- and high-J components result from H-roaming around CH3CO core and CH3-roaming around CHO radical, respectively, in consistency with a recent assignment by Kable and co-workers (Lee et al., Chem. Sci. 5, 4633 (2014)). The H-roaming pathway disappears at the CO(v ≥ 2) states, because of insufficient available energy following bond-breaking of H + CH3CO. By analyzing the CH4 emission spectrum, we obtained a bimodal vibrational distribution; the low-energy component is ascribed to the transition state (TS) pathway, consistent with prediction by quasiclassical trajectory calculations, while the high-energy component results from H- and CH3-roamings. A branching fraction of H-roaming/CH3-roaming/TS contribution is evaluated to be (8% ± 3%)/(68% ± 10%)/(25% ± 5%), in which the TS pathway was observed for the first time. The three pathways proceed concomitantly along the electronic ground state surface.

  10. Dengue Vectors and their Spatial Distribution.

    Science.gov (United States)

    Higa, Yukiko

    2011-12-01

    The distribution of dengue vectors, Ae. aegypti and Ae. albopictus, is affected by climatic factors. In addition, since their life cycles are well adapted to the human environment, environmental changes resulting from human activity such as urbanization exert a great impact on vector distribution. The different responses of Ae. aegypti and Ae albopictus to various environments result in a difference in spatial distribution along north-south and urban-rural gradients, and between the indoors and outdoors. In the north-south gradient, climate associated with survival is an important factor in spatial distribution. In the urban-rural gradient, different distribution reflects a difference in adult niches and is modified by geographic and human factors. The direct response of the two species to the environment around houses is related to different spatial distribution indoors and outdoors. Dengue viruses circulate mainly between human and vector mosquitoes, and the vector presence is a limiting factor of transmission. Therefore, spatial distribution of dengue vectors is a significant concern in the epidemiology of the disease.Current technologies such as GIS, satellite imagery and statistical models allow researchers to predict the spatial distribution of vectors in the changing environment. Although it is difficult to confirm the actual effect of environmental and climate changes on vector abundance and vector-borne diseases, environmental changes caused by humans and human behavioral changes due to climate change can be expected to exert an impact on dengue vectors. Longitudinal monitoring of dengue vectors and viruses is therefore necessary.

  11. Capsid Engineering of Adenovirus Vectors: Overcoming Early Vector-Host Interactions for Therapy.

    Science.gov (United States)

    Hagedorn, Claudia; Kreppel, Florian

    2017-10-01

    Adenovirus-based vectors comprise the most frequently used vector type in clinical studies to date. Both intense lab research and insights from the clinical trials reveal the importance of a comprehensive understanding of vector-host interactions. Especially for systemic intravenous adenovirus vector delivery, it is paramount to develop safe and efficacious vectors. Very early vector-host interactions that take place in blood long before the first cell is being transduced are phenomena triggered by the surface, shape, and size of the adenovirus vector particles. Not surprisingly, a multitude of different technologies ranging from genetics to chemistry has been developed to alter the adenovirus vector surface. In this review, we discuss the most important technologies and evaluate them for their suitability to overcome hurdles imposed by early vector-host interactions.

  12. Supporting medical decisions with vector decision trees.

    Science.gov (United States)

    Sprogar, M; Kokol, P; Zorman, M; Podgorelec, V; Yamamoto, R; Masuda, G; Sakamoto, N

    2001-01-01

    The article presents the extension of a common decision tree concept to a multidimensional - vector - decision tree constructed with the help of evolutionary techniques. In contrary to the common decision tree the vector decision tree can make more than just one suggestion per input sample. It has the functionality of many separate decision trees acting on a same set of training data and answering different questions. Vector decision tree is therefore simple in its form, is easy to use and analyse and can express some relationships between decisions not visible before. To explore and test the possibilities of this concept we developed a software tool--DecRain--for building vector decision trees using the ideas of evolutionary computing. Generated vector decision trees showed good results in comparison to classical decision trees. The concept of vector decision trees can be safely and effectively used in any decision making process.

  13. Vector assembly of colloids on monolayer substrates

    Science.gov (United States)

    Jiang, Lingxiang; Yang, Shenyu; Tsang, Boyce; Tu, Mei; Granick, Steve

    2017-06-01

    The key to spontaneous and directed assembly is to encode the desired assembly information to building blocks in a programmable and efficient way. In computer graphics, raster graphics encodes images on a single-pixel level, conferring fine details at the expense of large file sizes, whereas vector graphics encrypts shape information into vectors that allow small file sizes and operational transformations. Here, we adapt this raster/vector concept to a 2D colloidal system and realize `vector assembly' by manipulating particles on a colloidal monolayer substrate with optical tweezers. In contrast to raster assembly that assigns optical tweezers to each particle, vector assembly requires a minimal number of optical tweezers that allow operations like chain elongation and shortening. This vector approach enables simple uniform particles to form a vast collection of colloidal arenes and colloidenes, the spontaneous dissociation of which is achieved with precision and stage-by-stage complexity by simply removing the optical tweezers.

  14. [Spatial vector electrocardiography: technique, perspectives of use].

    Science.gov (United States)

    Bakutskiĭ, V N; Volobuev, A N; Kriukov, N N; Romanchuk, P I

    2003-01-01

    Potentials of the use of computer synthesis of integral electrical vector of the heart D0 are described. Calculation of spatial angular vector velocity and linear velocity of its movement along trajectory can be carried out in a framework of biophysical dipole model. Spatial presentation of vector is realized and its behavior in accordance with established pathologies discussed. Possible diagnostic value of obtained results and utility of their introduction into clinical practice are stressed.

  15. Genetic manipulation of endosymbionts to control vector and vector borne diseases

    Directory of Open Access Journals (Sweden)

    Jay Prakash Gupta

    Full Text Available Vector borne diseases (VBD are on the rise because of failure of the existing methods of control of vector and vector borne diseases and the climate change. A steep rise of VBDs are due to several factors like selection of insecticide resistant vector population, drug resistant parasite population and lack of effective vaccines against the VBDs. Environmental pollution, public health hazard and insecticide resistant vector population indicate that the insecticides are no longer a sustainable control method of vector and vector-borne diseases. Amongst the various alternative control strategies, symbiont based approach utilizing endosymbionts of arthropod vectors could be explored to control the vector and vector borne diseases. The endosymbiont population of arthropod vectors could be exploited in different ways viz., as a chemotherapeutic target, vaccine target for the control of vectors. Expression of molecules with antiparasitic activity by genetically transformed symbiotic bacteria of disease-transmitting arthropods may serve as a powerful approach to control certain arthropod-borne diseases. Genetic transformation of symbiotic bacteria of the arthropod vector to alter the vector’s ability to transmit pathogen is an alternative means of blocking the transmission of VBDs. In Indian scenario, where dengue, chikungunya, malaria and filariosis are prevalent, paratransgenic based approach can be used effectively. [Vet World 2012; 5(9.000: 571-576

  16. Sialic Acid Expression in the Mosquito Aedes aegypti and Its Possible Role in Dengue Virus-Vector Interactions

    Directory of Open Access Journals (Sweden)

    Jorge Cime-Castillo

    2015-01-01

    Full Text Available Dengue fever (DF is the most prevalent arthropod-borne viral disease which affects humans. DF is caused by the four dengue virus (DENV serotypes, which are transmitted to the host by the mosquito Aedes aegypti that has key roles in DENV infection, replication, and viral transmission (vector competence. Mosquito saliva also plays an important role during DENV transmission. In this study, we detected the presence of sialic acid (Sia in Aedes aegypti tissues, which may have an important role during DENV-vector competence. We also identified genome sequences encoding enzymes involved in Sia pathways. The cDNA for Aedes aegypti CMP-Sia synthase (CSAS was amplified, cloned, and functionally evaluated via the complementation of LEC29.Lec32 CSAS-deficient CHO cells. AedesCSAS-transfected LEC29.Lec32 cells were able to express Sia moieties on the cell surface. Sequences related to α-2,6-sialyltransferase were detected in the Aedes aegypti genome. Likewise, we identified Sia-α-2,6-DENV interactions in different mosquito tissues. In addition, we evaluated the possible role of sialylated molecules in a salivary gland extract during DENV internalization in mammalian cells. The knowledge of early DENV-host interactions could facilitate a better understanding of viral tropism and pathogenesis to allow the development of new strategies for controlling DENV transmission.

  17. Scalar Calibration of Vector Magnetometers

    DEFF Research Database (Denmark)

    Merayo, José M.G.; Brauer, Peter; Primdahl, Fritz

    2000-01-01

    The calibration parameters of a vector magnetometer are estimated only by the use of a scalar reference magnetometer. The method presented in this paper differs from those previously reported in its linearized parametrization. This allows the determination of three offsets or signals in the absence...... of a magnetic field, three scale factors for normalization of the axes and three non-orthogonality angles which build up an orthogonal system intrinsically in the sensor. The advantage of this method compared with others lies in its linear least squares estimator, which finds independently and uniquely...... the parameters for a given data set. Therefore, a magnetometer may be characterized inexpensively in the Earth's magnetic-field environment. This procedure has been used successfully in the pre-flight calibration of the state-of-the-art magnetometers on board the magnetic mapping satellites Orsted, Astrid-2...

  18. 3D vector flow imaging

    DEFF Research Database (Denmark)

    Pihl, Michael Johannes

    The main purpose of this PhD project is to develop an ultrasonic method for 3D vector flow imaging. The motivation is to advance the field of velocity estimation in ultrasound, which plays an important role in the clinic. The velocity of blood has components in all three spatial dimensions, yet...... Oscillation (TO) method, which estimates both the axial and the lateral velocity components. The first part of the scientific contribution demonstrates that a commercial implementation of the TO method is feasible. Afterwards, the method is expanded to a phased array geometry, and performance metrics based......:1 parallel receive beamforming. Simulation results demonstrate the feasibility of the method. In the final part, an experimental investigation of the 3D TO method is presented. Velocity measurements of steady flow were conducted in a flow-rig system, and the data were acquired using an experimental...

  19. An elusive vector dark matter

    Directory of Open Access Journals (Sweden)

    Chuan-Ren Chen

    2015-02-01

    Full Text Available Even though the sensitivity of direct dark matter search experiments reaches the level of about 10−45 cm2, no confident signal of dark matter has been observed. We point out that, if dark matter is a vector boson, the null result in direct dark matter search experiments may be due to the destructive effects in dark-matter–nucleon elastic scattering. We illustrate the scenario using a modified Higgs portal model that includes exotic quarks. The significant cancellation can occur for a certain mass gap between new heavy quark and dark matter. As a result, the spin-independent dark-matter–nucleon elastic scattering is so suppressed that the future direct search experiments will hardly observe the signal of dark matter.

  20. Endoplasmic Reticulum-Associated rht-PA Processing in CHO Cells: Influence of Mild Hypothermia and Specific Growth Rates in Batch and Chemostat Cultures.

    Directory of Open Access Journals (Sweden)

    Mauricio Vergara

    Full Text Available Chinese hamster ovary (CHO cells are the main host for producing recombinant proteins with human therapeutic applications mainly because of their capability to perform proper folding and glycosylation processes. In addition, mild hypothermia is one of the main strategies for maximising the productivity of these systems. However, little information is available on the effect of culture temperature on the folding and degradation processes of recombinant proteins that takes place in the endoplasmic reticulum.In order to evaluate the effect of the mild hypothermia on processing/endoplasmatic reticulum-associated degradation (ERAD processes, batch cultures of CHO cells producing recombinant human tissue plasminogen activator (rht-PA were carried out at two temperatures (37°C and 33°C and treated with specific inhibitors of glycosylation and ERAD I (Ubiquitin/Proteasome system or ERAD II (Autophagosoma/Lisosomal system pathways. The effect of mild hypothermia was analysed separately from its indirect effect on specific cell growth rate. To do this, chemostat cultures were carried out at the same incubation conditions as the batch cultures, controlling cell growth at high (0.017 h-1 and low (0.012 h-1 dilution rates. For a better understanding of the investigated phenomenon, cell behaviour was also analysed using principal component analysis (PCA.Results suggest that rht-PA is susceptible to degradation by both ERAD pathways studied, revealing that processing and/or ERAD processes are sensitive to temperature cultivation in batch culture. Moreover, by isolating the effect of culture temperature from the effect of cell growth rate verifyed by using chemostat cultures, we have found that processing and/or ERAD processes are more sensitive to reduction in specific growth rate than low temperature, and that temperature reduction may have a positive effect on protein processing. Interestingly, PCA indicated that the integrated performance displayed by CHO

  1. Identification of a Novel “Almost Neutral” Mu Opioid Receptor Antagonist in CHO Cells Expressing the Cloned Human Mu Opioid Receptor

    Science.gov (United States)

    Sally, Elliott J.; Xu, Heng; Dersch, Christina M.; Hsin, Ling-Wei; Chang, Li-Te; Prisinzano, Thomas E.; Simpson, Denise S.; Giuvelis, Denise; Rice, Kenner C.; Jacobson, Arthur E.; Cheng, Kejun; Bilsky, Edward J.; Rothman, Richard B.

    2009-01-01

    The basal (constitutive) activity of G protein-coupled receptors allows for the measurement of inverse agonist activity. Some competitive antagonists turn into inverse agonists under conditions where receptors are constitutively active. In contrast, neutral antagonists have no inverse agonist activity, and they block both agonist and inverse agonist activity. The mu opioid receptor (MOR) demonstrates detectable constitutive activity only after a state of dependence is produced by chronic treatment with a MOR agonist. We therefore sought to identify novel MOR inverse agonists, and novel neutral MOR antagonists in both untreated and agonist-treated MOR cells. CHO cells expressing the cloned human mu receptor (hMOR-CHO cells) were incubated for 20 hr with medium (control) or 10 μM (2S,4aR,6aR,7R,9S,10aS,10bR)-9-(benzoyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho-[2,1-c]pyran-7-carboxylic acid methyl ester (herkinorin, HERK). HERK-treatment generates a high degree of basal signaling and enhances the ability to detect inverse agonists. [35S]-GTP-γ-S assays were conducted using established methods. We screened 21 MOR “antagonists” using membranes prepared from HERK-treated hMOR-CHO cells. All antagonists, including CTAP and 6β-naltrexol, were inverse agonists. However, LTC-2 7 4 ( (-)-3-cyclopropylmethyl-2,3,4,4aα,5,6,7,7aα-octahydro-1H-benzofuro[3,2-e]isoquinolin-9-ol)) showed the lowest efficacy as an inverse agonist, and, at concentrations less than 5 nM, had minimal effects on basal [35S]-GTP-γ-S binding. Other efforts in this study identified KC-2-009 ((+)-3-((1R,5S)-2-((Z)-3-Phenylallyl)-2-azabicyclo[3.3.1]nonan-5-yl)phenol hydrochloride) as an inverse agonist at untreated MOR cells. In HERK-treated cells, KC-2-009 had the highest efficacy as an inverse agonist. In summary, we identified a novel and selective MOR inverse agonist (KC-2-009), and a novel MOR antagonist (LTC-274) that shows the least inverse agonist activity among 21

  2. Integrated miRNA, mRNA and protein expression analysis reveals the role of post-transcriptional regulation in controlling CHO cell growth rate

    Directory of Open Access Journals (Sweden)

    Clarke Colin

    2012-11-01

    Full Text Available Abstract Background To study the role of microRNA (miRNA in the regulation of Chinese hamster ovary (CHO cell growth, qPCR, microarray and quantitative LC-MS/MS analysis were utilised for simultaneous expression profiling of miRNA, mRNA and protein. The sample set under investigation consisted of clones with variable cellular growth rates derived from the same population. In addition to providing a systems level perspective on cell growth, the integration of multiple profiling datasets can facilitate the identification of non-seed miRNA targets, complement computational prediction tools and reduce false positive and false negative rates. Results 51 miRNAs were associated with increased growth rate (35 miRNAs upregulated and 16 miRNAs downregulated. Gene ontology (GO analysis of genes (n=432 and proteins (n=285 found to be differentially expressed (DE identified biological processes driving proliferation including mRNA processing and translation. To investigate the influence of miRNA on these processes we combined the proteomic and transcriptomic data into two groups. The first set contained candidates where evidence of translational repression was observed (n=158. The second group was a mixture of proteins and mRNAs where evidence of translational repression was less clear (n=515. The TargetScan algorithm was utilised to predict potential targets within these two groups for anti-correlated DE miRNAs. Conclusions The evidence presented in this study indicates that biological processes such as mRNA processing and protein synthesis are correlated with growth rate in CHO cells. Through the integration of expression data from multiple levels of the biological system a number of proteins central to these processes including several hnRNPs and components of the ribosome were found to be post-transcriptionally regulated. We utilised the expression data in conjunction with in-silico tools to identify potential miRNA-mediated regulation of m

  3. Vector optimization set-valued and variational analysis

    CERN Document Server

    Chen, Guang-ya; Yang, Xiaogi

    2005-01-01

    This book is devoted to vector or multiple criteria approaches in optimization. Topics covered include: vector optimization, vector variational inequalities, vector variational principles, vector minmax inequalities and vector equilibrium problems. In particular, problems with variable ordering relations and set-valued mappings are treated. The nonlinear scalarization method is extensively used throughout the book to deal with various vector-related problems. The results presented are original and should be interesting to researchers and graduates in applied mathematics and operations research

  4. A multi-pronged investigation into the effect of glucose starvation and culture duration on fed-batch CHO cell culture

    DEFF Research Database (Denmark)

    Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian

    2015-01-01

    In this study, omics-based analysis tools were used to explore the effect of glucose starvation and culture duration on monoclonal antibody (mAb) production in fed-batch CHO cell culture to gain better insight into how these parameters can be controlled to ensure optimal mAb productivity...... and quality. Titer and N-glycosylation of mAbs, as well as proteomic signature and metabolic status of the production cells in the culture were assessed. We found that the impact of glucose starvation on the titer and N-glycosylation of mAbs was dependent on the degree of starvation during early stationary...... to the interplay between the dilution effect associated with change in specific productivity of mAbs and the changed nucleotide sugar metabolism. Herein, we also show and discuss that increased cell culture duration negatively affect the maturation of glycans. In addition, comparative proteomics analysis of cells...

  5. Influence of incorporated bromodeoxyuridine on the induction of chromosomal alterations by ionizing radiation and long-wave UV in CHO cells.

    Science.gov (United States)

    Zwanenburg, T S; van Zeeland, A A; Natarajan, A T

    1985-01-01

    Incorporation of BrdUrd into nuclear DNA sensitizes CHO cells (1) to the induction of chromosomal aberrations by X-rays and 0.5 MeV neutrons and (2) to induction of chromosomal aberrations and SCEs by lw-UV. We have attempted to establish a correlation between induced chromosomal alterations and induced single- or double-strand breaks in DNA. The data show that while DSBs correlate very well with X-ray-induced aberrations, no clear correlation could be established between lw-UV induced SSBs (including alkali-labile sites) and chromosomal alterations. In addition the effect of 3-aminobenzamide (3AB) on the induction of chromosomal aberrations and SCEs induced by lw-UV has been determined. It is shown that 3AB is without any effect when lw-UV-irradiated cells are posttreated with this inhibitor. The significance of these results is discussed.

  6. Extremal vectors and rectifiability | Enflo | Quaestiones Mathematicae

    African Journals Online (AJOL)

    The concept of extremal vectors of a linear operator with a dense range but not onto on a Hilbert space was introduced by P. Enflo in 1996 as a new approach to study invariant subspaces. Following this, there were several studies on analytic and geometric properties of backward minimal vectors and their applications to ...

  7. Nonlinear dynamics of a vectored thrust aircraft

    DEFF Research Database (Denmark)

    Sørensen, C.B; Mosekilde, Erik

    1996-01-01

    With realistic relations for the aerodynamic coefficients, numerical simulations are applied to study the longitudional dynamics of a thrust vectored aircraft. As function of the thrust magnitude and the thrust vectoring angle the equilibrium state exhibits two saddle-node bifurcations and three...

  8. Clustering Categories in Support Vector Machines

    DEFF Research Database (Denmark)

    Carrizosa, Emilio; Nogales-Gómez, Amaya; Morales, Dolores Romero

    2017-01-01

    The support vector machine (SVM) is a state-of-the-art method in supervised classification. In this paper the Cluster Support Vector Machine (CLSVM) methodology is proposed with the aim to increase the sparsity of the SVM classifier in the presence of categorical features, leading to a gain in in...

  9. Semantic Vector Space Model: Implementation and Evaluation.

    Science.gov (United States)

    Liu, Geoffrey Z.

    1997-01-01

    Presents the Semantic Vector Space Model, a text representation and searching technique based on the combination of Vector Space Model with heuristic syntax parsing and distributed representation of semantic case structures. In this model, both documents and queries are represented as semantic matrices, and retrieval is achieved by computing…

  10. Construction of expression vectors carrying mouse peroxisomal ...

    African Journals Online (AJOL)

    pUcD3 eukaryotic expression vector to express tagged-PEP protein for transient transfection analysis and identifying intracellular localization of PEP protein in future experiments. PEP-cDNA was amplified in different PCR reactions using pEGFP-PEP vector and 2 sets of primers introducing specific restriction sites at the ...

  11. Radiative corrections to vector boson masses

    NARCIS (Netherlands)

    Veltman, M.J.G.

    1980-01-01

    Weak and e.m. radiative corrections to vector boson masses are computed. Including corrections due to the presently known leptons and quarks, mass shifts of+3080 and +3310 MeV are obtained for the masses of the charged and neutral vector boson.

  12. Deep Support Vector Machines for Regression Problems

    NARCIS (Netherlands)

    Wiering, Marco; Schutten, Marten; Millea, Adrian; Meijster, Arnold; Schomaker, Lambertus

    2013-01-01

    In this paper we describe a novel extension of the support vector machine, called the deep support vector machine (DSVM). The original SVM has a single layer with kernel functions and is therefore a shallow model. The DSVM can use an arbitrary number of layers, in which lower-level layers contain

  13. Malaria vector control: from past to future.

    Science.gov (United States)

    Raghavendra, Kamaraju; Barik, Tapan K; Reddy, B P Niranjan; Sharma, Poonam; Dash, Aditya P

    2011-04-01

    Malaria is one of the most common vector-borne diseases widespread in the tropical and subtropical regions. Despite considerable success of malaria control programs in the past, malaria still continues as a major public health problem in several countries. Vector control is an essential part for reducing malaria transmission and became less effective in recent years, due to many technical and administrative reasons, including poor or no adoption of alternative tools. Of the different strategies available for vector control, the most successful are indoor residual spraying and insecticide-treated nets (ITNs), including long-lasting ITNs and materials. Earlier DDT spray has shown spectacular success in decimating disease vectors but resulted in development of insecticide resistance, and to control the resistant mosquitoes, organophosphates, carbamates, and synthetic pyrethroids were introduced in indoor residual spraying with needed success but subsequently resulted in the development of widespread multiple insecticide resistance in vectors. Vector control in many countries still use insecticides in the absence of viable alternatives. Few developments for vector control, using ovitraps, space spray, biological control agents, etc., were encouraging when used in limited scale. Likewise, recent introduction of safer vector control agents, such as insect growth regulators, biocontrol agents, and natural plant products have yet to gain the needed scale of utility for vector control. Bacterial pesticides are promising and are effective in many countries. Environmental management has shown sufficient promise for vector control and disease management but still needs advocacy for inter-sectoral coordination and sometimes are very work-intensive. The more recent genetic manipulation and sterile insect techniques are under development and consideration for use in routine vector control and for these, standardized procedures and methods are available but need thorough

  14. Modulation de l'apoptose radioinduite par Ac-DEVD-CHO, un inhibiteur de protéases ``ice-like"

    Science.gov (United States)

    Weltin, D.; Holl, V.; Hyun, J. W.; Marchal, J.; Jung, G. M.; Dufour, P.; Bischoff, P.

    1998-04-01

    The “ICE-like" proteases, recently renamed caspases, are the human homologues of the Caenorhabditis elegans ced-3 gene product and are activated in the early steps of apoptosis. The aim of this work is to determine whether the inhibition of one of these proteases, namely caspase-3, is able to modify the cell sensitivity toward radiation-induced apoptosis. Murine spleen lymphocytes submitted to γ-radiations in presence of Ac-DVED-CHO, a caspase-3 specific inhibitor, exhibit a sharply reduced number of radiation-induced hypodiploid particules as compared to the controls and an almost total inhibition of the internucleosomal DNA fragmentation. However, both the anionic phospholipids externalisation, another specific hallmark of apoptosis, and the viability remain unchanged. Les protéases “ICE-like" ou caspases, sont les homologues humaines du produit du gène ced-3 du ver Caenorhabditis elegans et sont activées lors des étapes précoces de l'apoptose. L'objectif de ce travail vise à déterminer dans quelle mesure l'inhibition de l'une d'entre elles, la caspase-3 est susceptible de modifier la sensibilité des cellules vis-à-vis de l'apoptose radioinduite. Des lymphocytes spléniques murins irradiés en présence de Ac-DVED-CHO un inhibiteur spécifique de la caspase-3 présentent un taux de particules hypodiploïdes radioinduites bien inférieur à celui des contrôles et une diminution drastique de la fragmentation internucléosomale de l'ADN. Toutefois, ni l'externalisation des phospholipides anioniques, autre marqueur spécifique de l'apoptose, ni la viabilité ne sont affectées.

  15. Chlorination of Household Drinking Water among Cholera Patients' Households to Prevent Transmission of Toxigenic Vibrio cholerae in Dhaka, Bangladesh: CHoBI7 Trial

    Science.gov (United States)

    Rashid, Mahamud-ur; George, Christine Marie; Monira, Shirajum; Mahmud, Toslim; Rahman, Zillur; Mustafiz, Munshi; Saif-Ur-Rahman, K. M.; Parvin, Tahmina; Bhuyian, Sazzadul Islam; Zohura, Fatema; Begum, Farzana; Biswas, Shwapon Kumar; Akhter, Shamima; Zhang, Xiaotong; Sack, David; Sack, R. Bradley; Alam, Munirul

    2016-01-01

    Household members of cholera patients are at a 100 times higher risk of cholera infections than the general population because of shared contaminated drinking water sources and secondary transmission through poor household hygiene practices. In this study, we investigated the bactericidal concentration of free chlorine required to inactivate Vibrio cholerae in household drinking water in Dhaka, Bangladesh. In laboratory experiments, we found that the concentrations of free chlorine required to inactivate 105 colony-forming units (CFU)/mL of V. cholerae serogroups O1 and O139 were 0.1 mg/L and 0.2 mg/L, respectively. The concentration of free chlorine generated by a single chlorine tablet (sodium dichloroisocyanurate [33 mg]) after a 30-minute reaction time in a 10-L sealed vessel containing Dhaka city municipal supply water was 1.8 mg/L; and the concentration declined to 0.26 mg/L after 24 hours. In field measurements, water collected from 165 households enrolled in a randomized controlled trial (RCT) of a chlorine and handwashing with soap intervention (Cholera-Hospital-Based-Intervention-for-7-Days[CHoBI7]), we observed significantly higher free chlorine concentrations in the 82 intervention arm households (mean = 1.12 mg/L, standard deviation [SD] = 0.52, range = 0.07–2.6 mg/L) compared with the 83 control households (0.017 mg/L, SD = 0.01, range = 0–0.06 mg/L) (P water in households of cholera patients in Dhaka city. This result is consistent with the findings from the RCT of CHoBI7 which found that this intervention led to a significant reduction in symptomatic cholera infections among household members of cholera patients and no stored drinking water samples with detectable V. cholerae. PMID:27698273

  16. Development of a chemically defined platform fed-batch culture media for monoclonal antibody-producing CHO cell lines with optimized choline content.

    Science.gov (United States)

    Kuwae, Shinobu; Miyakawa, Ichiko; Doi, Tomohiro

    2018-01-11

    A chemically defined platform basal medium and feed media were developed using a single Chinese hamster ovary (CHO) cell line that produces a monoclonal antibody (mAb). Cell line A, which showed a peak viable cell density of 5.9 × 10 6  cells/mL and a final mAb titer of 0.5 g/L in batch culture, was selected for the platform media development. Stoichiometrically balanced feed media were developed using glucose as an indicator of cell metabolism to determine the feed rates of all other nutrients. A fed-batch culture of cell line A using the platform fed-batch medium yielded a 6.4 g/L mAb titer, which was 12-fold higher than that of the batch culture. To examine the applicability of the platform basal medium and feed media, three other cell lines (A16, B, and C) that produce mAbs were cultured using the platform fed-batch medium, and they yielded mAb titers of 8.4, 3.3, and 6.2 g/L, respectively. The peak viable cell densities of the three cell lines ranged from 1.3 × 10 7 to 1.8 × 10 7  cells/mL. These results show that the nutritionally balanced fed-batch medium and feeds worked well for other cell lines. During the medium development, we found that choline limitation caused a lower cell viability, a lower mAb titer, a higher mAb aggregate content, and a higher mannose-5 content. The optimal choline chloride to glucose ratio for the CHO cell fed-batch culture was determined. Our platform basal medium and feed media will shorten the medium-development time for mAb-producing cell lines.

  17. Insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells.

    Science.gov (United States)

    Mariati; Yeo, Jessna H M; Koh, Esther Y C; Ho, Steven C L; Yang, Yuansheng

    2014-01-01

    The human cytomegalovirus promoter (hCMV) is susceptible to gene silencing in CHO cells, most likely due to epigenetic events, such as DNA methylation and histone modifications. The core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene has been shown to prevent DNA methylation. A set of modified hCMV promoters was developed by inserting one or two copies of IE in either forward or reverse orientations either upstream of the hCMV enhancer, between the enhancer and core promoter (CP), or downstream of the CP. The modified hCMV with one copy of IE inserted between the enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability without compromising expression level when compared with the wild-type (WT) hCMV. A third of 18 EGFP expressing clones generated using MR1 retained 70% of their starting expression level after 8 weeks of culture in the absence of selection pressure, while none of 18 WT hCMV generated clones had expression above 50%. MR1 also improved antibody expression stability of methotrexate (MTX) amplified CHO cell lines. Stably transfected pools generated using MR1 maintained 62% of their original monoclonal antibody titer after 8 weeks of culture in the absence of MTX, compared to only 37% for WT hCMV pools. Low levels of CpG methylation within both WT hCMV and MR1 were observed in all the analyzed cell lines and the methylation levels did not correlate to the expression stability, suggesting IE enhances expression stability by other mechanisms other than preventing methylation. © 2014 American Institute of Chemical Engineers.

  18. Effects of 2.45-GHz Electromagnetic Fields with a Wide Range of SARs on Micronucleus Formation in CHO-K1 Cells

    Directory of Open Access Journals (Sweden)

    S. Koyama

    2004-01-01

    Full Text Available There has been considerable discussion about the influence of high-frequency electromagnetic fields (HFEMF on the human body. In particular, HFEMF used for mobile phones may be of great concern for human health. In order to investigate the properties of HFEMF, we have examined the effects of 2.45-GHz EMF on micronucleus (MN formation in Chinese hamster ovary (CHO-K1 cells. MN formation is induced by chromosomal breakage or inhibition of spindles during cell division and leads to cell damage. We also examined the influence of heat on MN formation, since HFEMF exposure causes a rise in temperature. CHO-K1 cells were exposed to HFEMF for 2 h at average specific absorption rates (SARs of 5, 10, 20, 50, 100, and 200 W/kg, and the effects on these cells were compared with those in sham-exposed control cells. The cells were also treated with bleomycin alone as a positive control or with combined treatment of HFEMF exposure and bleomycin. Heat treatment was performed at temperatures of 37, 38, 39, 40, 41, and 42°C.The MN frequency in cells exposed to HFEMF at a SAR of lower than 50 W/kg did not differ from the sham-exposed controls, while those at SARs of 100 and 200 W/kg were significantly higher when compared with the sham-exposed controls. There was no apparent combined effect of HFEMF exposure and bleomycin treatment. On heat treatment at temperatures from 38–42°C, the MN frequency increased in a temperature-dependent manner. We also showed that an increase in SAR causes a rise in temperature and this may be connected to the increase in MN formation generated by exposure to HFEMF.

  19. Evaluation of antimutagenic activity and mechanisms of action of beta-glucan from barley, in CHO-k1 and HTC cell lines using the micronucleus test.

    Science.gov (United States)

    Oliveira, Rodrigo Juliano; Ribeiro, Lúcia Regina; da Silva, Ariane Fernanda; Matuo, Renata; Mantovani, Mário Sérgio

    2006-10-01

    Due to the need to identify new antimutagenic agents and to determine their mechanism of action, the present study examined the mechanism of action of the beta-glucan with regard to antimutagenicity using the micronucleus assay in CHO-k1 and HTC cell lines. The mutagenicity experiments were performed with three different concentrations of beta-glucan (5, 10, and 20 microg/mL), in wich only the highest dose showed mutagenic activity. In the antimutagenicity experiments, the same concentrations of beta-glucan were combined with a mutagenic agent, methylmethane sulfonate, or 2-aminoanthracene, using four different treatment protocols: pre-treatment, simultaneous treatment (simple and with pre-incubation), and post-treatment. The results indicate that the CHO-k1 cell line treated with MMS presented a chemopreventive activity for all the doses of beta-glucan in the different treatment protocols, except for the lowest dose in post-treatment. When HTC cell line treated with MMS is analysed, a chemopreventive activity can be verified for the highest dose in both pre- and post-treatment. For the simple simultaneous treatment, the three doses demonstrated efficacy, while for the simultaneous treatment with pre-incubation only the intermediate concentration was effective. In HTC treated with 2AA both the lowest dose in the pre-treatment protocol and the post-treatment protocol did not show efficacy in preventing DNA damage. The evaluation of the different protocols and the damage decrease percentages observed suggest that beta-glucan has both desmutagenic and bioantimutagenic activity. It is necessary, however, to note that efficacy and mechanism of action are subject to variation when compared the two cell lines, since in HTC, representing a drug-metabolizing system, this substance can show a diminished chemopreventive capacity.

  20. ARP2, a novel pro-apoptotic protein expressed in epithelial prostate cancer LNCaP cells and epithelial ovary CHO transformed cells.

    Science.gov (United States)

    Mas-Oliva, Jaime; Navarro-Vidal, Enrique; Tapia-Vieyra, Juana Virginia

    2014-01-01

    Neoplastic epithelial cells generate the most aggressive types of cancers such as those located in the lung, breast, colon, prostate and ovary. During advanced stages of prostate cancer, epithelial cells are associated to the appearance of androgen-independent tumors, an apoptotic-resistant phenotype that ultimately overgrows and promotes metastatic events. We have previously identified and electrophysiologically characterized a novel Ca(2+)-permeable channel activated during apoptosis in the androgen-independent prostate epithelial cancer cell line, LNCaP. In addition, we reported for the first time the cloning and characterization of this channel-like molecule named apoptosis regulated protein 2 (ARP2) associated to a lethal influx of Ca(2+) in Xenopus oocytes. In the present study, LNCaP cells and Chinese hamster ovary cells (CHO cell line) transfected with arp2-cDNA are induced to undergo apoptosis showing an important impact on cell viability and activation of caspases 3 and 7 when compared to serum deprived grown cells and ionomycin treated cells. The subcellular localization of ARP2 in CHO cells undergoing apoptosis was studied using confocal microscopy. While apoptosis progresses, ARP2 initially localized in the peri-nuclear region of cells migrates with time towards the plasma membrane region. Based on the present results and those of our previous studies, the fact that ARP2 constitutes a novel cation channel is supported. Therefore, ARP2 becomes a valuable target to modulate the influx and concentration of calcium in the cytoplasm of epithelial cancer cells showing an apoptotic-resistant phenotype during the onset of an apoptotic event.

  1. Radiobiological response of U251MG, CHO-K1 and V79 cell lines to accelerator-based boron neutron capture therapy.

    Science.gov (United States)

    Sato, Eisuke; Zaboronok, Alexander; Yamamoto, Tetsuya; Nakai, Kei; Taskaev, Sergey; Volkova, Olga; Mechetina, Ludmila; Taranin, Alexander; Kanygin, Vladimir; Isobe, Tomonori; Mathis, Bryan J; Matsumura, Akira

    2017-12-21

    In the current article, we provide in vitro efficacy evaluation of a unique accelerator-based neutron source, constructed at the Budker Institute of Nuclear Physics (Novosibirsk, Russian Federation), for boron neutron capture therapy (BNCT), which is particularly effective in the case of invasive cancers. U251MG, CHO-K1 and V79 cells were incubated and irradiated in various concentrations of boric acid with epithermal neutrons for 2-3 h in a plexiglass phantom, using 2.0 MeV proton energy and 1.5-3.0 mA proton current, resulting in a neutron fluence of 2.16 × 1012 cm-2. The survival curves of cells loaded with boron were normalized to those irradiated without boron (to exclude the influence of the fast neutron and gamma dose components) and fit to the linear-quadratic (LQ) model. Colony formation assays showed the following cell survival rates (means ± SDs): CHO-K1: 0.348 ± 0.069 (10 ppm), 0.058 ± 0.017 (20 ppm), 0.018 ± 0.005 (40 ppm); V79: 0.476 ± 0.160 (10 ppm), 0.346 ± 0.053 (20 ppm), 0.078 ± 0.015 (40 ppm); and U251MG: 0.311 ± 0.061 (10 ppm), 0.131 ± 0.022 (20 ppm), 0.020 ± 0.010 (40 ppm). The difference between treated cells and controls was significant in all cases (P BNCT into the clinical phase. © The Author(s) 2017. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

  2. Axial vector Z‧ and anomaly cancellation

    Science.gov (United States)

    Ismail, Ahmed; Keung, Wai-Yee; Tsao, Kuo-Hsing; Unwin, James

    2017-05-01

    Whilst the prospect of new Z‧ gauge bosons with only axial couplings to the Standard Model (SM) fermions is widely discussed, examples of anomaly-free renormalisable models are lacking in the literature. We look to remedy this by constructing several motivated examples. Specifically, we consider axial vectors which couple universally to all SM fermions, as well as those which are generation-specific, leptophilic, and leptophobic. Anomaly cancellation typically requires the presence of new coloured and charged chiral fermions, and we argue that in a large class of models masses of these new states are expected to be comparable to that of the axial vector. Finally, an axial vector mediator could provide a portal between SM and hidden sector states, and we also consider the possibility that the axial vector couples to dark matter. If the dark matter relic density is set due to freeze-out via the axial vector, this strongly constrains the parameter space.

  3. Cascade Support Vector Machines with Dimensionality Reduction

    Directory of Open Access Journals (Sweden)

    Oliver Kramer

    2015-01-01

    Full Text Available Cascade support vector machines have been introduced as extension of classic support vector machines that allow a fast training on large data sets. In this work, we combine cascade support vector machines with dimensionality reduction based preprocessing. The cascade principle allows fast learning based on the division of the training set into subsets and the union of cascade learning results based on support vectors in each cascade level. The combination with dimensionality reduction as preprocessing results in a significant speedup, often without loss of classifier accuracies, while considering the high-dimensional pendants of the low-dimensional support vectors in each new cascade level. We analyze and compare various instantiations of dimensionality reduction preprocessing and cascade SVMs with principal component analysis, locally linear embedding, and isometric mapping. The experimental analysis on various artificial and real-world benchmark problems includes various cascade specific parameters like intermediate training set sizes and dimensionalities.

  4. Evaluation of the radio modifier effect of propolis on chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60-CO; Avaliacao do efeito radiomodificador da propolis em celulas de ovario de hamster chines (CHO-K1) e em celulas tumorais de prostata (PC3), irradiadas com CO-60

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Geyza Spigoti

    2011-07-01

    In the last decades, it has been given a great interest to investigations concerning natural, effective, nontoxic compounds with radioprotective potential together with the increasing utilization of different types of ionizing radiation for various applications. Among them propolis, a resinous compound produced by honeybees (Apis mellifera), has been considered quite promising, since it presents several advantageous biological characteristics, i. e., anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant and also free radical scavenging action. The purpose of the present study was to evaluate the effect of Brazilian propolis, collected in the State of Rio Grande do Sul, on Chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with {sup 60}Co {gamma} radiation. For this purpose, three interlinked parameters were analyzed: micronucleus induction, cell viability and clonogenic death. The choice of these parameters was justified by their biological significance, in addition to the fact that they are readily observable and measurable in irradiated cells. The cytogenetic data obtained showed a radioprotective effect of propolis (5-100 {mu}g/ml) in the induction of DNA damage for both cell lines, irradiated with doses of 1 - 4 Gy. The cytotoxicity assay, however, showed a prominent antiproliferative effect of propolis (50 - 400{mu}/ml) in PC3 cells irradiated with 5 G{gamma}. The survival curves obtained were adequately fitted by a linear-quadratic model, where the {alpha} coefficient was higher in CHO-K1 cells. Concerning the clonogenic capacity, PC3 cells were more radiosensitive than CHO-K1 cells at the higher doses of the survival curve. Propolis at the concentrations of 30 - 100 {mu}g/ml, did not influence the clonogenic potential of PC3 cells, since the survival curves, associated or not with propolis, were found similar, although the combined treatment in CHO-K1 cells exhibited a stimulating proliferative effect. The data

  5. Geoacoustic inversion using the vector field

    Science.gov (United States)

    Crocker, Steven E.

    The main goal of this project was to study the use of the acoustic vector field, separately or in combination with the scalar field, to estimate the depth dependent geoacoustic properties of the seafloor via non-linear inversion. The study was performed in the context of the Sediment Acoustics Experiment 2004 (SAX04) conducted in the Northern Gulf of Mexico (GOM) where a small number of acoustic vector sensors were deployed in close proximity to the seafloor. A variety of acoustic waveforms were transmitted into the seafloor at normal incidence. The acoustic vector sensors were located both above and beneath the seafloor interface where they measured the acoustic pressure and the acoustic particle acceleration. Motion data provided by the buried vector sensors were affected by a suspension response that was sensitive to the mass properties of the sensor, the sediment density and sediment elasticity (e.g., shear wave speed). The suspension response for the buried vector sensors included a resonance within the analysis band of 0.4 to 2.0 kHz. The suspension resonance represented an unknown complex transfer function between the acoustic vector field in the seabed and data representing that field. Therefore, inverse methods developed for this study were required to 1) estimate dynamic properties of the sensor suspension resonance and 2) account for the associated corruption of vector field data. A method to account for the vector sensor suspense response function was integrated directly into the inversion methods such that vector channel data corruption was reduced and an estimate of the shear wave speed in the sediment was returned. Inversions of real and synthetic data sets indicated that information about sediment shear wave speed was carried by the suspension response of the buried sensors, as opposed to being contained inherently within the acoustic vector field.

  6. Biosafety of onco-retroviral vectors.

    Science.gov (United States)

    VandenDriessche, Thierry; Collen, Désiré; Chuah, Marinee K L

    2003-12-01

    Extensive gene therapy studies in preclinical models and in clinical trials underscore the relative safety of onco-retroviral vectors. Up until recently, no adverse effects have been reported in nearly 2000 patients that were enrolled in gene therapy clinical trials involving onco-retroviral vectors. However, the main safety concern of using onco-retroviral vectors is related to the risk of malignant transformation following oncogene activation due to random onco-retroviral genomic integration. Based on primate studies, there is an apparent low risk of malignancy that is predominately associated with the occurrence of chronic retroviremia resulting from replication-competent retroviruses (RCR), particularly in immunosuppressed recipient hosts. However, in the latest packaging cell lines and vectors, the risk of RCR-generation has been drastically reduced, primarily by minimizing the homologous overlap between vector and helper sequences. Nevertheless, results from a recent preclinical study in mice and a clinical trial in patients suffering from SCID-X1 strongly suggest that onco-retroviral vectors devoid of RCR can contribute to lymphomagenesis by insertional activation of cellular oncogenes. The risk of inadvertent germline transmission of onco-retroviral vectors appears to be low, especially relative to the endogenous rate of germline insertion, which is known to occur naturally in the human population via transmission of endogenous retro-transposons. The strict dependency of onco-retroviral gene transfer on cell division is an important safety advantage that significantly limits the risks of horizontal transmission. Since improved onco-retroviral vectors or transduction protocols may result in an increased number of retroviral integrations per cell, this may concomitantly increase the risk of malignant transformation. The use of suicide genes, self-inactivating vectors and/or chromosomal insulators is, therefore, warranted to further enhance the safety features

  7. Inactivation of GDP-fucose transporter gene (Slc35c1) in CHO cells by ZFNs, TALENs and CRISPR-Cas9 for production of fucose-free antibodies.

    Science.gov (United States)

    Chan, Kah Fai; Shahreel, Wahyu; Wan, Corrine; Teo, Gavin; Hayati, Noor; Tay, Shi Jie; Tong, Wen Han; Yang, Yuansheng; Rudd, Pauline M; Zhang, Peiqing; Song, Zhiwei

    2016-03-01

    Removal of core fucose from N-glycans attached to human IgG1 significantly enhances its affinity for the receptor FcγRIII and thereby dramatically improves its antibody-dependent cellular cytotoxicity activity. While previous works have shown that inactivation of fucosyltransferase 8 results in mutants capable of producing fucose-free antibodies, we report here the use of genome editing techniques, namely ZFNs, TALENs and the CRISPR-Cas9, to inactivate the GDP-fucose transporter (SLC35C1) in Chinese hamster ovary (CHO) cells. A FACS approach coupled with a fucose-specific lectin was developed to rapidly isolate SLC35C1-deficient cells. Mass spectrometry analysis showed that both EPO-Fc produced in mutants arising from CHO-K1 and anti-Her2 antibody produced in mutants arising from a pre-existing antibody-producing CHO-HER line lacked core fucose. Lack of functional SLC35C1 in these cells does not affect cell growth or antibody productivity. Our data demonstrate that inactivating Slc35c1 gene represents an alternative approach to generate CHO cells for production of fucose-free antibodies. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. An analysis of the dispute process regarding high-level nuclear waste repository siting in Toyo-cho, Japan: Decisive factors in the dispute and roles of the governments and experts

    Energy Technology Data Exchange (ETDEWEB)

    Komatsuzaki, Shunsaku; Horii, Hideyuki (Dept. of Civil Engineering, Univ. of Tokyo (Japan)); Saigo, Takahiro (Mitsubishi Research Institute, Inc. (Japan))

    2010-09-15

    The siting policy of HLW repository in Japan was 'application-based' until 2007 and Toyo-cho is the only municipality which applied for the Literature Survey. In Toyo-cho, however, a serious antagonism among citizens occurred and the application was withdrawn after the mayor was replaced by election. Our detailed analysis of the process based on the methods of political science and psychology shows five decisive factors: 1) opposing activists both in the town and from outside successfully changed citizens' perceptions of HLW by rhetorical expressions, 2) the mayor lacks careful actions and effective policy adviser, 3) NUMO, an organization which runs HLW projects, didn't effectively coordinate Toyo-cho and stakeholders, 4) the municipal government and council exercised very limited influences on the dispute despite their political authority, and 5) the existence of grant adversely influenced the citizens since it causes criticism that Toyo-cho applies a repository for grant. We finally conclude that the substantial problems, caused by the five decisive factors, were the propagation of enthusiastic opposition and the lack of peaceful deliberation based on local governance. In order to avoid enthusiastic opposition and to realize responsible decision making, or negotiation, we suggest that A) active and prompt response of experts, especially political/administrative ones, to radical opposing activities, B) solution to the adverse influence of the grant by the government's agenda

  9. Vector 33: A reduce program for vector algebra and calculus in orthogonal curvilinear coordinates

    Science.gov (United States)

    Harper, David

    1989-06-01

    This paper describes a package with enables REDUCE 3.3 to perform algebra and calculus operations upon vectors. Basic algebraic operations between vectors and between scalars and vectors are provided, including scalar (dot) product and vector (cross) product. The vector differential operators curl, divergence, gradient and Laplacian are also defined, and are valid in any orthogonal curvilinear coordinate system. The package is written in RLISP to allow algebra and calculus to be performed using notation identical to that for operations. Scalars and vectors can be mixed quite freely in the same expression. The package will be of interest to mathematicians, engineers and scientists who need to perform vector calculations in orthogonal curvilinear coordinates.

  10. GRASS GIS Vector Processing: Towards GRASS 7

    Science.gov (United States)

    Metz, Markus; Landa, Martin; Petrasova, Anna; Petras, Vaclav; Chemin, Yann; Neteler, Markus

    2014-05-01

    The upcoming GRASS GIS 7 release improves not only raster processing and general design but the vector processing in the first place. GRASS GIS, as a topological GIS, recognizes that the topology plays the key role in the vector processing and analysis. Topology ensures that adjacent geographic components in a single vector map are related. In contrast to non-topological GIS, a border common to two areas exists only once and is shared between the two areas. Topological representation of vector data helps to produce and maintain vector maps with clean geometry as well as enables the user to perform certain analyses that can not be conducted with non-topological or spaghetti data. Non-topological vector data are automatically converted to a topological representation upon import. Further more, various cleaning tools exist to remove non-trivial topological errors. In the upcoming GRASS GIS 7 release the vector library was particularly improved to make it faster and more efficient with an improved internal vector file format. This new topological format reduces memory and disk space requirements, leading to a generally faster processing. Opening an existing vector requires less memory providing additionally support for large files. The new spatial index performs queries faster (compared to GRASS GIS 6 more than 10 times for large vectors). As a new option the user can select a file-based version of the spatial index for large vector data. All topological cleaning tools have been optimized with regard to processing speed, robustness, and system requirements. The topological engine comes with a new prototype for direct read/write support of Simple Features API/OGR. Additionally vector data can be directly exchanged with topological PostGIS 2 databases. Considering the wide spread usage of ESRI Shapefile, a non-topological format for vector data exchange, it is particularly advantageous that GRASS GIS 7 offers advanced cleaning tools. For power users and programmers, the

  11. Correlated Topic Vector for Scene Classification.

    Science.gov (United States)

    Wei, Pengxu; Qin, Fei; Wan, Fang; Zhu, Yi; Jiao, Jianbin; Ye, Qixiang

    2017-07-01

    Scene images usually involve semantic correlations, particularly when considering large-scale image data sets. This paper proposes a novel generative image representation, correlated topic vector, to model such semantic correlations. Oriented from the correlated topic model, correlated topic vector intends to naturally utilize the correlations among topics, which are seldom considered in the conventional feature encoding, e.g., Fisher vector, but do exist in scene images. It is expected that the involvement of correlations can increase the discriminative capability of the learned generative model and consequently improve the recognition accuracy. Incorporated with the Fisher kernel method, correlated topic vector inherits the advantages of Fisher vector. The contributions to the topics of visual words have been further employed by incorporating the Fisher kernel framework to indicate the differences among scenes. Combined with the deep convolutional neural network (CNN) features and Gibbs sampling solution, correlated topic vector shows great potential when processing large-scale and complex scene image data sets. Experiments on two scene image data sets demonstrate that correlated topic vector improves significantly the deep CNN features, and outperforms existing Fisher kernel-based features.

  12. HVJ liposomes and HVJ envelope vectors.

    Science.gov (United States)

    Kaneda, Yasufumi

    2011-10-01

    This protocol describes techniques for construction of fusion-mediated vectors based on inactivated HVJ (hemagglutinating virus of Japan; Sendai virus). HVJ liposomes are constructed by fusing liposomes containing DNA with inactivated HVJ. The HVJ envelope vector, a more simplified vector, incorporates DNA into inactivated HVJ particles without liposomes. Both vectors have many advantages. They can be used to introduce proteins, peptides, oligonucleotides (including antisense oligonucleotides, decoy oligonucleotides, and ribozymes), and short interfering RNA (siRNA), as well as plasmid DNA, into cultured cells in vitro and into organs in vivo. Fusion-mediated delivery avoids the degradation of therapeutic molecules before reaching the cytoplasm. Finally, repeated injection of the vector in vivo is not inhibited and even enhances the effects of the delivered molecules. These vectors have been used in many gene therapy experiments in animal models to address problems such as liver cirrhosis, hearing impairment, ischemic brain damage, peripheral arterial diseases, and cancers. This protocol describes methods for the preparation of HVJ liposomes and of HVJ envelope vectors and their use in delivery of plasmid DNA into various cells and tissues.

  13. Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

    Directory of Open Access Journals (Sweden)

    Marcela Cristina Correa de Freitas

    2014-06-01

    Full Text Available OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO and baby hamster kidney cells (BHK. Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K.METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones.RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293.CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.

  14. Malaria vector control and personal protection.

    Science.gov (United States)

    2006-01-01

    Malaria transmission rates and risks can be greatly reduced by vector control, mitigating high malaria incidence and prevalence rates. Methods and strategies for malaria vector control (MVC) have been well documented by WHO, although its implementation varies widely. Technical guidelines for MVC strategies and materials are readily available, but the status and role of MVC have not been reviewed and redefined in terms of programme management and resource allocation. There are huge changes since November 1993 when the last WHO Study Group reviewed vector control for malaria and other mosquito-borne diseases, following the 1992 adoption of the Global Malaria Control Strategy. Operationally, with reform of the health sector in many countries, the centrally managed and vertically structured malaria control programme (MCP) has been superseded by a community-based and decentralized one. This poses challenges for effective implementation of MVC strategies. Therefore it became evident that the role of vector control in malaria control needs to be reconsidered to develop a strategic framework for MVC implementation by national malaria control programmes and other partners. This report of a WHO Study Group on Malaria Vector Control and Personal Protection reviewed the current vector control strategies and their effectiveness in various operational and eco-epidemiological settings, and identified challenges for implementation in different health systems. An outline strategic framework for strengthening malaria vector control implementation was developed. The process of deciding about which mosquito control method is appropriate in a given situation should be guided by an analysis of the level of malaria endemicity and vector bionomics, the eco-epidemiological setting, the health management system and an estimate of the programme sustainability. This report also provides a basis for the development of a strategic framework for strengthening malaria vector control

  15. Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains.

    Science.gov (United States)

    Balabashin, Dmitriy; Kovalenko, Elena; Toporova, Viktoria; Aliev, Teimur; Panina, Anna; Svirshchevskaya, Elena; Dolgikh, Dmitry; Kirpichnikov, Mikhail

    2015-10-01

    Tumor necrosis factor-α (TNF-α) plays a key role in rheumatoid arthritis and some other autoimmune diseases. Therapy with anti-TNF-α recombinant antibodies (Ab) appears to be highly effective. Production of new hyper-producing eukaryotic cell lines can decrease the treatment cost, which currently is very high. However, due to the complexity of protein transcription, translation, processing, and secretion in mammalian cells, the stages at which antibody expression is affected are still poorly determined. The aim of this work was to compare the productivity of two cell lines developed in CHO DG44 cells, deficient in dihydrofolate reductase, transfected with vectors carrying either heavy (H) or light (L) chains of chimeric antibody under different combinations of selective elements. Both H and L chains were cloned either in pOptiVEC or pcDNA3.3 vectors and different combinations were used to produce HL and LH cell lines. We have shown that Ab production has been low and comparable between HL and LH cells until selection on methotrexate (MTX) when LH but not HL cells have responded with 3.5 times increased productivity. Flow cytometry analysis has demonstrated that intracellular concentration of full size Abs in LH cells was 5.6 times higher than in HL ones due to higher amount of H chain synthesis. No differences in viability between HL and LH cells have been found. We have concluded that the expression of H chain in the pOptiVEC vector, which is responsible for MTX resistance, has led to the suppression of H chain synthesis and limitation in full Ab assembly.

  16. Meromorphic Vector Fields and Circle Packings

    DEFF Research Database (Denmark)

    Dias, Kealey

    of dynamical systems, continuous and discrete, correspond to the study of differential equations (vector fields) and iteration of mappings respectively. In holomorphic dynamics, the systems studied are restricted to those described by holomorphic (complex analytic) functions or meromorphic (allowing poles...... problems. Since the class of complex polynomial vector fields in the plane is natural to consider, it is remarkable that its study has only begun very recently. There are numerous fundamental questions that are still open, both in the general classification of these vector fields, the decomposition...

  17. Vector analysis for mathematicians, scientists and engineers

    CERN Document Server

    Simons, S

    1970-01-01

    Vector Analysis for Mathematicians, Scientists and Engineers, Second Edition, provides an understanding of the methods of vector algebra and calculus to the extent that the student will readily follow those works which make use of them, and further, will be able to employ them himself in his own branch of science. New concepts and methods introduced are illustrated by examples drawn from fields with which the student is familiar, and a large number of both worked and unworked exercises are provided. The book begins with an introduction to vectors, covering their representation, addition, geome

  18. Interior point decoding for linear vector channels

    Energy Technology Data Exchange (ETDEWEB)

    Wadayama, T [Nagoya Institute of Technology, Gokiso, Showa-ku, Nagoya, Aichi, 466-8555 (Japan)], E-mail: wadayama@nitech.ac.jp

    2008-01-15

    In this paper, a novel decoding algorithm for low-density parity-check (LDPC) codes based on convex optimization is presented. The decoding algorithm, called interior point decoding, is designed for linear vector channels. The linear vector channels include many practically important channels such as inter-symbol interference channels and partial response channels. It is shown that the maximum likelihood decoding (MLD) rule for a linear vector channel can be relaxed to a convex optimization problem, which is called a relaxed MLD problem.

  19. Some remarks on a generalized vector product

    OpenAIRE

    Primitivo Acosta-Humánez; Moisés Aranda; Reinaldo Núñez

    2011-01-01

    In this paper we use a generalized vector product to construct an exterior form ∧ : (Rn) k → R( n k), where n k = n! (n−k)!k! , k ≤ n. Finally, for n = k − 1 we introduce the reversing operation to study this generalized vector product over palindromic and antipalindromic vectors Resumen. En este artículo usamos un producto vectorial generalizado para construir una forma exterior ∧ : (Rn) k → R( n k), en donde como es natural, n k = n! ...

  20. Gene therapy using retrovirus vectors: vector development and biosafety at clinical trials.

    Science.gov (United States)

    Doi, Knayo; Takeuchi, Yasuhiro

    2015-01-01

    Retrovirus vectors (gammaretroviral and lentiviral vectors) have been considered as promising tools to transfer therapeutic genes into patient cells because they can permanently integrate into host cellular genome. To treat monogenic, inherited diseases, retroviral vectors have been used to add correct genes into patient cells. Conventional gammaretroviral vectors achieved successful results in clinical trials: treated patients had therapeutic gene expression in target cells and had improved symptoms of diseases. However, serious side-effects of leukemia occurred, caused by retroviral insertional mutagenesis (IM). These incidences stressed the importance of monitoring vector integration sites in patient cells as well as of re-consideration on safer vectors. More recently lentiviral vectors which can deliver genes into non-dividing cells started to be used in clinical trials including neurological disorders, showing their efficacy. Vector integration site analysis revealed that lentiviruses integrate less likely to near promoter regions of oncogenes than gammaretroviruses and no adverse events have been reported in lentiviral vector-mediated gene therapy clinical trials. Therefore lentiviral vectors have promises to be applied to a wide range of common diseases in near future. For example, T cells from cancer patients were transduced to express chimeric T cell receptors recognizing their tumour cells enhancing patients' anti-cancer immunity.

  1. Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy

    OpenAIRE

    Wold, William S. M.; Toth, Karoly

    2013-01-01

    Adenovirus vectors are the most commonly employed vector for cancer gene therapy. They are also used for gene therapy and as vaccines to express foreign antigens. Adenovirus vectors can be replication-defective; certain essential viral genes are deleted and replaced by a cassette that expresses a foreign therapeutic gene. Such vectors are used for gene therapy, as vaccines, and for cancer therapy. Replication-competent (oncolytic) vectors are employed for cancer gene therapy. Oncolytic vector...

  2. Taxonomy and Biology of Phlebotomine Vectors of Human Disease.

    Science.gov (United States)

    DIPTERA, *DISEASE VECTORS, *TAXONOMY, *VECTOR ANALYSIS , ANTIBODIES, BIOLOGY , BRAIN, CHROMOSOMES, COLOMBIA, COLONIES( BIOLOGY ), DISEASES, FEMALES... HUMANS , INFECTIOUS DISEASES, LABORATORIES, LEISHMANIA, LEISHMANIASIS, NEUTRALIZATION, PATIENTS, SEROLOGY, STRAINS( BIOLOGY ).

  3. Dengue Vectors and their Spatial Distribution

    National Research Council Canada - National Science Library

    Higa, Yukiko

    2011-01-01

    .... In addition, since their life cycles are well adapted to the human environment, environmental changes resulting from human activity such as urbanization exert a great impact on vector distribution...

  4. Regional Disease Vector Ecology Profile: The Koreas

    Science.gov (United States)

    2015-01-01

    Similarly, the civilian population may experience displacement, homelessness , reduced sanitation, increased exposure to vectors and disease, and...visit either of the following websites: 1. The WHO Searchable Snake/Antivenin Database 2. The Toxinology website ( Australia based) 69 REFERENCES

  5. Viruses vector control proposal: genus Aedes emphasis

    Directory of Open Access Journals (Sweden)

    Nelson Nogueira Reis

    Full Text Available Abstract The dengue fever is a major public health problem in the world. In Brazil, in 2015, there were 1,534,932 cases, being 20,320 cases of severe form, and 811 deaths related to this disease. The distribution of Aedes aegypti, the vector, is extensive. Recently, Zika and Chikungunya viruses had arisen, sharing the same vector as dengue and became a huge public health issue. Without specific treatment, it is urgently required as an effective vector control. This article is focused on reviewing vector control strategies, their effectiveness, viability and economical impact. Among all, the Sterile Insect Technique is highlighted as the best option to be adopted in Brazil, once it is largely effectively used in the USA and Mexico for plagues related to agribusiness.

  6. Viral vector-based influenza vaccines

    Science.gov (United States)

    de Vries, Rory D.; Rimmelzwaan, Guus F.

    2016-01-01

    ABSTRACT Antigenic drift of seasonal influenza viruses and the occasional introduction of influenza viruses of novel subtypes into the human population complicate the timely production of effective vaccines that antigenically match the virus strains that cause epidemic or pandemic outbreaks. The development of game-changing vaccines that induce broadly protective immunity against a wide variety of influenza viruses is an unmet need, in which recombinant viral vectors may provide. Use of viral vectors allows the delivery of any influenza virus antigen, or derivative thereof, to the immune system, resulting in the optimal induction of virus-specific B- and T-cell responses against this antigen of choice. This systematic review discusses results obtained with vectored influenza virus vaccines and advantages and disadvantages of the currently available viral vectors. PMID:27455345

  7. Viruses vector control proposal: genus Aedes emphasis

    Directory of Open Access Journals (Sweden)

    Nelson Nogueira Reis

    2017-07-01

    Full Text Available The dengue fever is a major public health problem in the world. In Brazil, in 2015, there were 1,534,932 cases, being 20,320 cases of severe form, and 811 deaths related to this disease. The distribution of Aedes aegypti, the vector, is extensive. Recently, Zika and Chikungunya viruses had arisen, sharing the same vector as dengue and became a huge public health issue. Without specific treatment, it is urgently required as an effective vector control. This article is focused on reviewing vector control strategies, their effectiveness, viability and economical impact. Among all, the Sterile Insect Technique is highlighted as the best option to be adopted in Brazil, once it is largely effectively used in the USA and Mexico for plagues related to agribusiness.

  8. Low Power, Self Calibrated Vector Magnetometer Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This Phase I SBIR project investigates a novel approach to vector magnetometry based on high precision measurements of the total magnetic field. The calibration is...

  9. Inflight parity vector compensation for FDI

    Science.gov (United States)

    Hall, S. R.; Motyka, P.; Gai, E.; Deyst, J. J., Jr.

    The performance of a failure detection and isolation (FDI) algorithm applied to a redundant strapdown inertial measurement unit (IMU) is limited by sensor errors such as input axis misalignment, scale factor errors, and biases. This paper presents a technique for improving the performance of FDI algorithms applied to redundant strapdown IMUs. A Kalman filter provides estimates of those linear combinations of sensor errors that affect the parity vector. These estimates are used to form a compensated parity vector which does not include the effects of sensor errors. The compensated parity vector is then used in place of the uncompensated parity vector to make FDI decisions. Simulation results are presented in which the algorithm is tested in a realistic flight environment that includes vehicle maneuvers, the effects of turbulence, and sensor failures. The results show that the algorithm can significantly improve FDI performance, especially during vehicle maneuvers.

  10. Vector optimization theory, applications, and extensions

    CERN Document Server

    Jahn, Johannes

    2011-01-01

    This new edition of a key monograph has fresh sections on the work of Edgeworth and Pareto in its presentation in a general setting of the fundamentals and important results of vector optimization. It examines background material, applications and theories.

  11. Sensorless vector and direct torque control

    CERN Document Server

    Vas, Peter

    1998-01-01

    This is the first comprehensive book on sensorless high performance a.c. drives. It is essential reading for anyone interested in acquiring a solid background on sensorless torque-controlled drives. It presents a detailed and unified treatment of sensorless vector-controlled and direct-torque controlled drive systems. It also discusses the applications of artificial intelligence to drives. Where possible, space vector theory is used and emphasis is laid on detailed mathematical and physical analysis. Sensorless drive schemes for different types of permanent magnet synchronous motors, synchronous reluctance motors, and induction motors are also presented. These include more than twenty vector drives e.g. five types of MRAS-based vector drives, and eleven types of direct-torque-controlled (DTC) drives, e.g. the ABB DTC drive. However, torque-controlled switched reluctance motor drives are also discussed due to their emerging importance. The book also covers various drive applications using artificial intellige...

  12. Weaving Knotted Vector Fields with Tunable Helicity

    Science.gov (United States)

    Kedia, Hridesh; Foster, David; Dennis, Mark R.; Irvine, William T. M.

    2016-12-01

    We present a general construction of divergence-free knotted vector fields from complex scalar fields, whose closed field lines encode many kinds of knots and links, including torus knots, their cables, the figure-8 knot, and its generalizations. As finite-energy physical fields, they represent initial states for fields such as the magnetic field in a plasma, or the vorticity field in a fluid. We give a systematic procedure for calculating the vector potential, starting from complex scalar functions with knotted zero filaments, thus enabling an explicit computation of the helicity of these knotted fields. The construction can be used to generate isolated knotted flux tubes, filled by knots encoded in the lines of the vector field. Lastly, we give examples of manifestly knotted vector fields with vanishing helicity. Our results provide building blocks for analytical models and simulations alike.

  13. Insects as vectors: systematics and biology.

    Science.gov (United States)

    Rodhain, F

    2015-04-01

    Among the many complex relationships between insects and microorganisms such as viruses, bacteria and parasites, some have resulted in the establishment of biological systems within which the insects act as a biological vector for infectious agents. It is therefore advisable to understand the identity and biology of these vectors in depth, in order to define procedures for epidemiological surveillance and anti-vector control. The following are successively reviewed in this article: Anoplura (lice), Siphonaptera (fleas), Heteroptera (bugs: Cimicidae, Triatoma, Belostomatidae), Psychodidae (sandflies), Simuliidae (black flies), Ceratopogonidae (biting midges), Culicidae (mosquitoes), Tabanidae (horseflies) and Muscidae (tsetse flies, stable flies and pupipara). The authors provide a rapid overview of the morphology, systematics, development cycle and bio-ecology of each of these groups of vectors. Finally, their medical and veterinary importance is briefly reviewed.

  14. Some remarks on a generalized vector product

    Directory of Open Access Journals (Sweden)

    Primitivo Acosta-Humánez

    2011-12-01

    Full Text Available In this paper we use a generalized vector product to construct an exterior form ∧ : (Rn k → R( n k, where n k = n! (n−k!k! , k ≤ n. Finally, for n = k − 1 we introduce the reversing operation to study this generalized vector product over palindromic and antipalindromic vectors Resumen. En este artículo usamos un producto vectorial generalizado para construir una forma exterior ∧ : (Rn k → R( n k, en donde como es natural, n k = n! (n−k!k! , k ≤ n. Finalmente, para n = k − 1 introducimos la operación reversar para estudiar este producto vectorial generalizado sobre vectores palindrómicos y antipalindrómicos.

  15. Weaving Knotted Vector Fields with Tunable Helicity.

    Science.gov (United States)

    Kedia, Hridesh; Foster, David; Dennis, Mark R; Irvine, William T M

    2016-12-30

    We present a general construction of divergence-free knotted vector fields from complex scalar fields, whose closed field lines encode many kinds of knots and links, including torus knots, their cables, the figure-8 knot, and its generalizations. As finite-energy physical fields, they represent initial states for fields such as the magnetic field in a plasma, or the vorticity field in a fluid. We give a systematic procedure for calculating the vector potential, starting from complex scalar functions with knotted zero filaments, thus enabling an explicit computation of the helicity of these knotted fields. The construction can be used to generate isolated knotted flux tubes, filled by knots encoded in the lines of the vector field. Lastly, we give examples of manifestly knotted vector fields with vanishing helicity. Our results provide building blocks for analytical models and simulations alike.

  16. MAGELLAN SURFACE CHARACTERISTICS VECTOR DATA RECORD

    Data.gov (United States)

    National Aeronautics and Space Administration — This data set contains the Magellan Surface Characteristics Vector Data Record (SCVDR) which is an orbit-by-orbit reduction of Magellan scattering and emission...

  17. VEST: Abstract vector calculus simplification in Mathematica

    Science.gov (United States)

    Squire, J.; Burby, J.; Qin, H.

    2014-01-01

    We present a new package, VEST (Vector Einstein Summation Tools), that performs abstract vector calculus computations in Mathematica. Through the use of index notation, VEST is able to reduce three-dimensional scalar and vector expressions of a very general type to a well defined standard form. In addition, utilizing properties of the Levi-Civita symbol, the program can derive types of multi-term vector identities that are not recognized by reduction, subsequently applying these to simplify large expressions. In a companion paper Burby et al. (2013) [12], we employ VEST in the automation of the calculation of high-order Lagrangians for the single particle guiding center system in plasma physics, a computation which illustrates its ability to handle very large expressions. VEST has been designed to be simple and intuitive to use, both for basic checking of work and more involved computations.

  18. Application of Vector Triggering Random Decrement

    DEFF Research Database (Denmark)

    Asmussen, J. C.; Ibrahim, S. R.; Brincker, Rune

    1997-01-01

    result is a Random Decrement function from each measurement. In traditional Random Decrement estimation the triggering condition is a scalar condition, which should only be fulfilled in a single measurement. In vector triggering Random Decrement the triggering condition is a vector condition....... The advantage of this new approach should be a reduction in estimation time without a significant loss of accuracy, since the vector triggering conditions ensure cross information between the measurements in the Random Decrement functions. The different problems with this technique is highlighted in two......This paper deals with applications of the vector triggering Random Decrement technique. This technique is new and developed with the aim of minimizing estimation time and identification errors. The theory behind the technique is discussed in an accompanying paper. The results presented...

  19. Application of Vector Triggering Random Decrement

    DEFF Research Database (Denmark)

    Asmussen, J. C.; Ibrahim, S. R.; Brincker, Rune

    result is a Random Decrement function from each measurement. In traditional Random Decrement estimation the triggering condition is a scalar condition, which should only be fulfilled in a single measurement. In vector triggering Random Decrement the triggering condition is a vector condition....... The advantage of this new approach should be a reduction in estimation time without a significant loss of accuracy, since the vector triggering conditions ensure cross information between the measurements in the Random Decrement functions. The different problems with this technique is highlighted in two......This paper deals with applications of the vector triggering Random Decrement technique. This technique is new and developed with the aim of minimizing estimation time and identification errors. The theory behind the technique is discussed in an accompanying paper. The results presented...

  20. Free divisors in prehomogeneous vector spaces

    OpenAIRE

    Granger, Michel; Mond, David; Schulze, Mathias

    2009-01-01

    We study linear free divisors, that is, free divisors arising as discriminants in prehomogeneous vector spaces, and in particular in quiver representation spaces. We give a characterization of the prehomogeneous vector spaces containing such linear free divisors. For reductive linear free divisors, we prove that the numbers of geometric and representation theoretic irreducible components coincide. As a consequence, we find that a quiver can only give rise to a linear free divisor if it has no...

  1. Acoustical vector solitons in anisotropic media

    Science.gov (United States)

    Adamashvili, G. T.; Peikrishvili, M. D.; Koplatadze, R. R.

    2017-04-01

    A theory of acoustical vector solitons of self-induced transparency in anisotropic media is developed. It is shown that, in these systems, a two-component vector soliton oscillating with the difference and sum of the frequencies in the vicinity of the frequency of a carrying acoustic wave may arise. Explicit analytical expressions for the form and parameters of a nonlinear wave depending on the direction of pulse propagation are given.

  2. Trichophoromyia auraensis is a putative vector

    Directory of Open Access Journals (Sweden)

    Carolina Bioni Garcia Teles

    Full Text Available The sandfly Trichophoromyia auraensis has recently evolved as a proven vector of Leishmania (Viannia endemic to state of Acre in the north of Brazil. This note is intended to propose a correction in the report of the first occurrence of natural infection of Leishmania (Viannia in this species. We and the other scientific groups reinforced that Tr. auraensis is a possible vector involved in the transmission of American cutaneous leishmaniasis in Acre, Brazil.

  3. Engineering HSV-1 vectors for gene therapy.

    Science.gov (United States)

    Goins, William F; Huang, Shaohua; Cohen, Justus B; Glorioso, Joseph C

    2014-01-01

    Virus vectors have been employed as gene transfer vehicles for various preclinical and clinical gene therapy applications, and with the approval of Glybera (alipogene tiparvovec) as the first gene therapy product as a standard medical treatment (Yla-Herttuala, Mol Ther 20: 1831-1832, 2013), gene therapy has reached the status of being a part of standard patient care. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing tumor cells have been used in Phase I-III human trials in patients with glioblastoma multiforme, a fatal form of brain cancer, and in malignant melanoma. In fact, T-VEC (talimogene laherparepvec, formerly known as OncoVex GM-CSF) displayed efficacy in a recent Phase III trial when compared to standard GM-CSF treatment alone (Andtbacka et al. J Clin Oncol 31: sLBA9008, 2013) and may soon become the second FDA-approved gene therapy product used in standard patient care. In addition to the replication-competent oncolytic HSV vectors like T-VEC, replication-defective HSV vectors have been employed in Phase I-II human trials and have been explored as delivery vehicles for disorders such as pain, neuropathy, and other neurodegenerative conditions. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are totally replication defective, nontoxic, and capable of long-term transgene expression in neurons. This chapter describes methods for the construction of recombinant genomic HSV vectors based on the HSV-1 replication-defective vector backbones, steps in their purification, and their small-scale production for use in cell culture experiments as well as preclinical animal studies.

  4. Effect of pathogen-resistant vectors on the transmission dynamics of a vector-borne disease.

    Science.gov (United States)

    Arino, Julien; Bowman, Chris; Gumel, Abba; Portet, Stéphanie

    2007-10-01

    A model is introduced for the transmission dynamics of a vector-borne disease with two vector strains, one wild and one pathogen-resistant; resistance comes at the cost of reduced reproductive fitness. The model, which assumes that vector reproduction can lead to the transmission or loss of resistance (reversion), is analyzed in a particular case with specified forms for the birth and force of infection functions. The vector component can have, in the absence of disease, a coexistence equilibrium where both strains survive. In the case where reversion is possible, this coexistence equilibrium is globally asymptotically stable when it exists. This equilibrium is still present in the full vector-host system, leading to a reduction of the associated reproduction number, thereby making elimination of the disease more feasible. When reversion is not possible, there can exist an additional equilibrium with only resistant vectors.

  5. Effects of Climate and Climate Change on Vectors and Vector-Borne Diseases: Ticks Are Different.

    Science.gov (United States)

    Ogden, Nick H; Lindsay, L Robbin

    2016-08-01

    There has been considerable debate as to whether global risk from vector-borne diseases will be impacted by climate change. This has focussed on important mosquito-borne diseases that are transmitted by the vectors from infected to uninfected humans. However, this debate has mostly ignored the biological diversity of vectors and vector-borne diseases. Here, we review how climate and climate change may impact those most divergent of arthropod disease vector groups: multivoltine insects and hard-bodied (ixodid) ticks. We contrast features of the life cycles and behaviour of these arthropods, and how weather, climate, and climate change may have very different impacts on the spatiotemporal occurrence and abundance of vectors, and the pathogens they transmit. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  6. Blocking transmission of vector-borne diseases.

    Science.gov (United States)

    Schorderet-Weber, Sandra; Noack, Sandra; Selzer, Paul M; Kaminsky, Ronald

    2017-04-01

    Vector-borne diseases are responsible for significant health problems in humans, as well as in companion and farm animals. Killing the vectors with ectoparasitic drugs before they have the opportunity to pass on their pathogens could be the ideal way to prevent vector borne diseases. Blocking of transmission might work when transmission is delayed during blood meal, as often happens in ticks. The recently described systemic isoxazolines have been shown to successfully prevent disease transmission under conditions of delayed pathogen transfer. However, if the pathogen is transmitted immediately at bite as it is the case with most insects, blocking transmission becomes only possible if ectoparasiticides prevent the vector from landing on or, at least, from biting the host. Chemical entities exhibiting repellent activity in addition to fast killing, like pyrethroids, could prevent pathogen transmission even in cases of immediate transfer. Successful blocking depends on effective action in the context of the extremely diverse life-cycles of vectors and vector-borne pathogens of medical and veterinary importance which are summarized in this review. This complexity leads to important parameters to consider for ectoparasiticide research and when considering the ideal drug profile for preventing disease transmission. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. Feature Vector Construction Method for IRIS Recognition

    Science.gov (United States)

    Odinokikh, G.; Fartukov, A.; Korobkin, M.; Yoo, J.

    2017-05-01

    One of the basic stages of iris recognition pipeline is iris feature vector construction procedure. The procedure represents the extraction of iris texture information relevant to its subsequent comparison. Thorough investigation of feature vectors obtained from iris showed that not all the vector elements are equally relevant. There are two characteristics which determine the vector element utility: fragility and discriminability. Conventional iris feature extraction methods consider the concept of fragility as the feature vector instability without respect to the nature of such instability appearance. This work separates sources of the instability into natural and encodinginduced which helps deeply investigate each source of instability independently. According to the separation concept, a novel approach of iris feature vector construction is proposed. The approach consists of two steps: iris feature extraction using Gabor filtering with optimal parameters and quantization with separated preliminary optimized fragility thresholds. The proposed method has been tested on two different datasets of iris images captured under changing environmental conditions. The testing results show that the proposed method surpasses all the methods considered as a prior art by recognition accuracy on both datasets.

  8. Black holes in vector-tensor theories

    Science.gov (United States)

    Heisenberg, Lavinia; Kase, Ryotaro; Minamitsuji, Masato; Tsujikawa, Shinji

    2017-08-01

    We study static and spherically symmetric black hole (BH) solutions in second-order generalized Proca theories with nonminimal vector field derivative couplings to the Ricci scalar, the Einstein tensor, and the double dual Riemann tensor. We find concrete Lagrangians which give rise to exact BH solutions by imposing two conditions of the two identical metric components and the constant norm of the vector field. These exact solutions are described by either Reissner-Nordström (RN), stealth Schwarzschild, or extremal RN solutions with a non-trivial longitudinal mode of the vector field. We then numerically construct BH solutions without imposing these conditions. For cubic and quartic Lagrangians with power-law couplings which encompass vector Galileons as the specific cases, we show the existence of BH solutions with the difference between two non-trivial metric components. The quintic-order power-law couplings do not give rise to non-trivial BH solutions regular throughout the horizon exterior. The sixth-order and intrinsic vector-mode couplings can lead to BH solutions with a secondary hair. For all the solutions, the vector field is regular at least at the future or past horizon. The deviation from General Relativity induced by the Proca hair can be potentially tested by future measurements of gravitational waves in the nonlinear regime of gravity.

  9. Auto-tuning Dense Vector and Matrix-vector Operations for Fermi GPUs

    DEFF Research Database (Denmark)

    Sørensen, Hans Henrik Brandenborg

    2012-01-01

    In this paper, we consider the automatic performance tuning of dense vector and matrix-vector operations on GPUs. Such operations form the backbone of level 1 and level 2 routines in the Basic Linear Algebra Subroutines (BLAS) library and are therefore of great importance in many scientific...... for dense vector and matrix-vector operations by appropriately utilizing the fine-grained parallelism of the GPU. Our tuned kernels display between 25-100% better performance than the current CUBLAS 3.2 library....

  10. In vitro genotoxic and cytotoxic effects of ivermectin and its formulation ivomec on Chinese hamster ovary (CHO{sub K1}) cells

    Energy Technology Data Exchange (ETDEWEB)

    Molinari, G.; Soloneski, S.; Reigosa, M.A. [Catedra de Citologia, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, La Plata (Argentina); Larramendy, M.L., E-mail: m_larramendy@hotmail.com [Catedra de Citologia, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, La Plata (Argentina)

    2009-06-15

    The effects of ivermectin (IVM) and its commercial formulation ivomec (IVM 1.0%) were studied on Chinese hamster ovary (CHO{sub K1}) cells by several genotoxicity [sister chromatid exchange (SCE) and single cell gel electrophoresis (SCGE)] and cytotoxicity [cell-cycle progression (CCP), mitotic index (MI), proliferative replication index (PRI), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and neutral red (NR)] bioassays within the 1.0-250 {mu}g/ml concentration-range. While IVM and ivomec did not modified SCE frequencies, they induced DNA-strand breaks revealed by SCGE. An enhancement of slightly damaged cells and a decrease in undamaged cells were observed in IVM-treated cultures with 5.0-50.0 {mu}g/ml. In ivomec-treated cells, while an increase in slightly damaged cells was induced with 5.0-50.0 {mu}g/ml, the damaged and undamaged cells increased and decreased only with 50.0 {mu}g/ml. Both compounds exerted a delay in CCP and a reduction in PRI when 25.0 {mu}g/ml was employed whereas cytotoxicity was observed at higher concentration than 50.0 {mu}g/ml. No MI alteration was observed with 1.0-10.0 and 1.0-5.0 {mu}g/ml of IVM and ivomec, respectively. A concentration-related trend to an increase in MI was achieved within 1.0-10.0 {mu}g/ml. An increase in the MI was induced in 10.0 {mu}g/ml ivomec-treated cultures. A marked reduction of about 89% and 62% in regard to controls was observed with 25.0 {mu}g/ml of IVM and ivomec, respectively. NR and MTT assays revealed a cell growth inhibition when 0.25-250.0 {mu}g/ml of both compounds was employed. The results highlighted that IVM and ivomec exert both genotoxicity and cytotoxicity in mammalian cells in vitro, at least in CHO{sub K1} cells.

  11. Lactobacillus gasseri [corrected] CHO-220 and inulin reduced plasma total cholesterol and low-density lipoprotein cholesterol via alteration of lipid transporters.

    Science.gov (United States)

    Ooi, L-G; Ahmad, R; Yuen, K-H; Liong, M-T

    2010-11-01

    This randomized, double-blind, placebo-controlled, and parallel-designed study was conducted to investigate the effect of a synbiotic product containing Lactobacillus gasseri [corrected] CHO-220 and inulin on lipid profiles of hypercholesterolemic men and women. Thirty-two hypercholesterolemic men and women with initial mean plasma cholesterol levels of 5.7±0.32 mmol/L were recruited for the 12-wk study. The subjects were randomly allocated to 2 groups; namely the treatment group (synbiotic product) and the control group (placebo), and each received 4 capsules of synbiotic or placebo daily. Our results showed that the mean body weight, energy, and nutrient intake of the subjects did not differ between the 2 groups over the study period. The supplementation of synbiotic reduced plasma total cholesterol and low-density lipoprotein (LDL)-cholesterol by 7.84 and 9.27%, respectively, compared with the control over 12 wk. Lipoproteins were subsequently subfractionated and characterized. The synbiotic supplementation resulted in a lower concentration of triglycerides in the very low, intermediate, low, and high-density lipoprotein particles compared with the control over 12 wk. The concentration of triglycerides in lipoproteins is positively correlated with an increased risk of atherosclerosis. Our results showed that the synbiotic might exhibit an atheropreventive characteristic. Cholesteryl ester (CE) in the high-density lipoprotein particles of the synbiotic group was also higher compared with the control, indicating greater transport of cholesterol in the form of CE to the liver for hydrolysis. This may have led to the reduced plasma total cholesterol level of the synbiotic group. The supplementation of synbiotic also reduced the concentration of CE in the LDL particles compared with the control, leading to the formation of smaller and denser particles that are more easily removed from blood. This supported the reduced LDL-cholesterol level of the synbiotic group

  12. Role of the leucine-rich domain of platelet GPIbalpha in correct post-translational processing--the Nancy I Bernard-Soulier mutation expressed on CHO cells.

    Science.gov (United States)

    Ulsemer, P; Lanza, F; Baas, M J; Schwartz, A; Ravanat, C; Briquel, M E; Cranmer, S; Jackson, S; Cazenave, J P; de la Salle, C

    2000-07-01

    The mechanisms governing the biosynthesis and surface expression of platelet adhesive receptors on parent megakaryocytes are as yet poorly understood. In particular, the assembly and processing of the multisubunit glycoprotein (GP) Ib-IX-V complex, a receptor for von Willebrand factor (vWf) is not fully understood. In the present work, these questions were addressed by reproducing a natural mutation of GPIbalpha found in a variant case of Bernard-Soulier syndrome (Nancy I), due to the deletion of leucine 179 in the seventh leucine-rich repeat of the polypeptide. Wild type and mutated GPIbalpha were transfected into CHO cells expressing GPlbbeta and GPIX. Flow cytometry showed surface expression of the three subunits of both GPIb-IX complexes, but GPlbalphadeltaLeu was present at lower levels (20-40%) and was recognized only by a sub class of monoclonal antibodies which epitopes were not modified by the mutation. These properties reproduce the defect found in the patient's platelets, demonstrating the causative nature of the mutation and validate the use of the CHO cells model. Biochemical studies were performed in an attempt to elucidate the mechanism of the conformational change of GPIbalphadeltaLeu. They unexpectedly revealed a major glycosylation deficiency of the mutated GPIbalpha leading to a 40% decrease in molecular weight. The other two subunits of the complex were however normal and present at the plasma membrane. The deletion led to complete functional deficiency with lack of vWf binding of CHOalphadeltaLeu transfected cells in the presence of botrocetin and defective adhesion to a vWf coated surface under static conditions. Finally, in contrast to normal CHOalphabetaIX cells, which displayed rolling and deceleration when perfused over a vWf surface, CHOalphadeltaLeubetaIX cells were unable to roll over or attach to a vWf substratum. These results show that the integrity of the leucine-rich region of GPIbalpha is essential for normal processing and

  13. Viral Vectors for in Vivo Gene Transfer

    Science.gov (United States)

    Thévenot, E.; Dufour, N.; Déglon, N.

    The transfer of DNA into the nucleus of a eukaryotic cell (gene transfer) is a central theme of modern biology. The transfer is said to be somatic when it refers to non-germline organs of a developed individual, and germline when it concerns gametes or the fertilised egg of an animal, with the aim of transmitting the relevant genetic modification to its descendents [1]. The efficient introduction of genetic material into a somatic or germline cell and the control of its expression over time have led to major advances in understanding how genes work in vivo, i.e., in living organisms (functional genomics), but also to the development of innovative therapeutic methods (gene therapy). The efficiency of gene transfer is conditioned by the vehicle used, called the vector. Desirable features for a vector are as follows: Easy to produce high titer stocks of the vector in a reproducible way. Absence of toxicity related to transduction (transfer of genetic material into the target cell, and its expression there) and no immune reaction of the organism against the vector and/or therapeutic protein. Stability in the expression of the relevant gene over time, and the possibility of regulation, e.g., to control expression of the therapeutic protein on the physiological level, or to end expression at the end of treatment. Transduction of quiescent cells should be as efficient as transduction of dividing cells. Vectors currently used fall into two categories: non-viral and viral vectors. In non-viral vectors, the DNA is complexed with polymers, lipids, or cationic detergents (described in Chap. 3). These vectors have a low risk of toxicity and immune reaction. However, they are less efficient in vivo than viral vectors when it comes to the number of cells transduced and long-term transgene expression. (Naked DNA transfer or electroporation is rather inefficient in the organism. This type of gene transfer will not be discussed here, and the interested reader is referred to the

  14. New Approach to Fractal Approximation of Vector-Functions

    OpenAIRE

    Konstantin Igudesman; Marsel Davletbaev; Gleb Shabernev

    2014-01-01

    This paper introduces new approach to approximation of continuous vector-functions and vector sequences by fractal interpolation vector-functions which are multidimensional generalization of fractal interpolation functions. Best values of fractal interpolation vector-functions parameters are found. We give schemes of approximation of some sets of data and consider examples of approximation of smooth curves with different conditions.

  15. Combined metabolomics and proteomics reveals hypoxia as a cause of lower productivity on scale-up to a 5000-liter CHO bioprocess.

    Science.gov (United States)

    Gao, Yuanwei; Ray, Somak; Dai, Shujia; Ivanov, Alexander R; Abu-Absi, Nicholas R; Lewis, Amanda M; Huang, Zhuangrong; Xing, Zizhuo; Borys, Michael C; Li, Zheng Jian; Karger, Barry L

    2016-09-01

    Large-scale bioprocessing is key to the successful manufacturing of a biopharmaceutical. However, cell viability and productivity are often lower in the scale-up from laboratory to production. In this study, we analyzed CHO cells, which showed lower percent viabilities and productivity in a 5-KL production scale bioreactor compared to a 20-L bench-top scale under seemingly identical process parameters. An increase in copper concentration in the media from 0.02 µM to 0.4 µM led to a doubling of percent viability in the production scale albeit still at a lower level than the bench-top scale. Combined metabolomics and proteomics revealed the increased copper reduced the presence of reactive oxygen species (ROS) in the 5-KL scale process. The reduction in oxidative stress was supported by the increased level of glutathione peroxidase in the lower copper level condition. The excess ROS was shown to be due to hypoxia (intermittent), as evidenced by the reduction in fibronectin with increased copper. The 20-L scale showed much less hypoxia and thus less excess ROS generation, resulting in little to no impact to productivity with the increased copper in the media. The study illustrates the power of 'Omics in aiding in the understanding of biological processes in biopharmaceutical production. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Assessment of cytotoxic and cytogenetic effects of a 1,2,5-thiadiazole derivative on CHO-K1 cells. Its application as corrosion inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Grillo, C.A. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA, CCT La Plata-CONICET), Facultad de Ciencias Exactas, Departamento de Quimica, Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4, 1900 La Plata (Argentina); Mirifico, M.V. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA, CCT La Plata-CONICET), Facultad de Ciencias Exactas, Departamento de Quimica, Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4, 1900 La Plata (Argentina); Facultad de Ingenieria, Areas Departamentales Ingenieria Quimica and Mecanica, Universidad Nacional de La Plata, Calle 47 y 1, 1900 La Plata (Argentina); Morales, M.L. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA, CCT La Plata-CONICET), Facultad de Ciencias Exactas, Departamento de Quimica, Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4, 1900 La Plata (Argentina); Reigosa, M.A. [IMBICE (Instituto Multidisciplinario de Biologia Celular), CICPBA, CONICET, Calle 526 entre 10 y 11, 1900 La Plata (Argentina); Mele, M. Fernandez Lorenzo de, E-mail: mmele@inifta.unlp.edu.ar [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA, CCT La Plata-CONICET), Facultad de Ciencias Exactas, Departamento de Quimica, Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4, 1900 La Plata (Argentina); Facultad de Ingenieria, Areas Departamentales Ingenieria Quimica and Mecanica, Universidad Nacional de La Plata, Calle 47 y 1, 1900 La Plata (Argentina)

    2009-10-30

    This work focuses on the possible use of phenanthro[9,10-c]-1,2,5-thiadiazole 1,1-dioxide (TDZ) as a harmless corrosion inhibitor. TDZ range-dose providing minimum adverse effects to the environment and human health, with satisfactory corrosion-inhibiting properties was evaluated. Cytotoxicity and genotoxicity of TDZ at 0.57-12.50 {mu}M concentration range were tested by neutral red, chromosomal aberrations, mitotic index, and colony formation assays. Results showed a significant increase of chromatid-type aberrations for the highest concentration of TDZ assayed (12.50 {mu}M). Additionally, a reduction in the proliferative rate for lower concentrations was detected by the MI assay. We concluded that TDZ should be used at concentrations lower than 1.16 {mu}M. Corrosion assays performed showed good inhibition effect (ca. 50%) at low (0.65 {mu}M) TDZ concentration. Consequently, our results indicated that TDZ induced a time- and dose-dependent genotoxic and cytotoxic response on CHO-K1 cells. Short assays should be complemented with long exposure tests to simulate chronic contact with TDZ since lower threshold levels may be found for shorter exposures and a wrong safety range could be determined.

  17. On-line monitoring of responses to nutrient feed additions by multi-frequency permittivity measurements in fed-batch cultivations of CHO cells.

    Science.gov (United States)

    Ansorge, Sven; Esteban, Geoffrey; Schmid, Georg

    2010-04-01

    Changes in the nutrient availability of mammalian cell cultures are reflected in the beta-dispersion parameter characteristic frequency (f ( C )) and the on-line dual frequency permittivity signal. Multi-frequency permittivity measurements were therefore evaluated in fed-batch cultivations of two different CHO cell lines. Similar responses to nutrient depletions and discontinuous feed additions were monitored in different cultivation phases and experimental setups. Sudden increases in permittivity and f ( C ) occurred when feed additions were conducted. A constant or declining permittivity value in combination with a decrease in f ( C ) indicated nutrient limitations. f ( C ) correlated well with changes in oxygen uptake rate when cell diameter remained constant, indicating that metabolic activity is reflected in the value of f ( C ). When significant cell size changes occurred during the cultivations, the analysis of the beta-dispersion parameters was rendered complex. For the application of our findings in other systems it will be hence required to conduct additional off-line measurements. Based on these results, it is hypothesized that multi-frequency permittivity measurements can give information on the intracellular or physiological state in fed-batch mode. Similar observations were made when using different cell lines and feeding strategies, indicating that the findings are transferable to other cell lines and systems. The results should lead to an improved understanding of routine fed-batch processes. Additional studies are, however, required to explore how these observations can be used for fed-batch process development and optimization.

  18. Combining mechanistic and data-driven approaches to gain process knowledge on the control of the metabolic shift to lactate uptake in a fed-batch CHO process.

    Science.gov (United States)

    Zalai, Dénes; Koczka, Krisztina; Párta, László; Wechselberger, Patrick; Klein, Tobias; Herwig, Christoph

    2015-01-01

    A growing body of knowledge is available on the cellular regulation of overflow metabolism in mammalian hosts of recombinant protein production. However, to develop strategies to control the regulation of overflow metabolism in cell culture processes, the effect of process parameters on metabolism has to be well understood. In this study, we investigated the effect of pH and temperature shift timing on lactate metabolism in a fed-batch Chinese hamster ovary (CHO) process by using a Design of Experiments (DoE) approach. The metabolic switch to lactate consumption was controlled in a broad range by the proper timing of pH and temperature shifts. To extract process knowledge from the large experimental dataset, we proposed a novel methodological concept and demonstrated its usefulness with the analysis of lactate metabolism. Time-resolved metabolic flux analysis and PLS-R VIP were combined to assess the correlation of lactate metabolism and the activity of the major intracellular pathways. Whereas the switch to lactate uptake was mainly triggered by the decrease in the glycolytic flux, lactate uptake was correlated to TCA activity in the last days of the cultivation. These metabolic interactions were visualized on simple mechanistic plots to facilitate the interpretation of the results. Taken together, the combination of knowledge-based mechanistic modeling and data-driven multivariate analysis delivered valuable insights into the metabolic control of lactate production and has proven to be a powerful tool for the analysis of large metabolic datasets. © 2015 American Institute of Chemical Engineers.

  19. Detection of genotoxicity of water from an urbanized stream, in Corbicula fluminea (Mollusca) (in vivo) and CHO-K1 cells (in vitro) using comet assay.

    Science.gov (United States)

    Rigonato, Janaina; Mantovani, Mário Sérgio; Jordão, Berenice Quinzani

    2010-07-01

    The comet assay was utilized to investigate the quality of water from seven locations along the Cambé Stream, in vivo (Corbicula fluminea hemolymph), in vitro (CHO-K1 cells), in situ, and in laboratory studies. The Cambé Stream basin (Londrina, PR, Brazil) is almost completely urbanized and receives different forms of industrial and domestic runoff. The data indicated the occurrence of DNA damage in cells examined in vivo and in vitro, shown by the significant increase in frequencies of cells with DNA damage after exposure to water from all seven locations used in the study. Our results strongly suggest the presence of genotoxic agent(s) in water at all of the sampled locations, demonstrated by elevated numbers of cells with DNA damaged in field and laboratory tests. In all of the places sampled, domestic sewage influence appeared to be one important cause for the introduction of xenobiotics, environmental genotoxins, and pollutants into the water. Thus, the comet assay applied in these cell systems was able to detect adverse environmental conditions, proving to be a very adequate short-term test and should be included in batteries of tests utilized in the monitoring of aquatic environments.

  20. Manipulation of the sodium-potassium ratio as a lever for controlling cell growth and improving cell specific productivity in perfusion CHO cell cultures.

    Science.gov (United States)

    Wang, Samantha B; Lee-Goldman, Alexandria; Ravikrishnan, Janani; Zheng, Lili; Lin, Henry

    2017-12-26

    Perfusion processes typically require removal of a continuous or semi-continuous volume of cell culture in order to maintain a desired target cell density. For fast growing cell lines, the product loss from this stream can be upwards of 35%, significantly reducing the overall process yield. As volume removed is directly proportional to cell growth, the ability to modulate growth during perfusion cell culture production thus becomes crucial. Leveraging existing media components to achieve such control without introducing additional supplements is most desirable because it decreases process complexity and eliminates safety and clearance concerns. Here, the impact of extracellular concentrations of sodium (Na) and potassium (K) on cell growth and productivity is explored. High throughput small-scale models of perfusion revealed Na:K ratios below 1 can significantly suppress cell growth by inducing cell cycle arrest in the G0/1 phase. A concomitant increase in cell specific productivity was also observed, reaching as high as 115 pg/cell/day for one cell line studied. Multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrated similar responses to lower Na:K media, indicating the universal applicability of such an approach. Product quality attributes were also assessed and revealed that effects were cell line specific, and can be acceptable or manageable depending on the phase of the drug development. Drastically altering Na and K levels in perfusion media as a lever to impact cell growth and productivity is proposed. © 2017 Wiley Periodicals, Inc.

  1. Linear antenna microwave plasma CVD diamond deposition at the edge of no-growth region of C-H-O ternary diagram

    Energy Technology Data Exchange (ETDEWEB)

    Potocky, Stepan; Babchenko, Oleg; Hruska, Karel; Kromka, Alexander [Institute of Physics AS CR, v.v.i., Cukrovarnicka 10, 16200 Praha (Czech Republic)

    2012-12-15

    The process parametric window for diamond deposition using the chemical vapor deposition at low pressures is quite limited where addition of oxygen in the gas phase broadens this window. The lower boundary of the lens-shaped domain in C-H-O ternary diagram concurs with the H{sub 2}-CO tie-line (C/(C + O) = 0.5). In this work, we present the set of experiments where the ratio of C/(C + O) was kept at a constant value 0.385. The effect of hydrogen concentration (ratio O/(O + H) varied from 0.047 to 0.364) on plasma characteristics and deposited NCD films were investigated. Raman spectroscopy confirmed the diamond character of all deposited coatings while scanning electron microscopy showed transformation from not closed to continuous film and further decrease of grain size and finally growth of diamond nanowires while decreasing hydrogen concentration in a gas mixture. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  2. The systematic study of the electroporation and electrofusion of B16-F1 and CHO cells in isotonic and hypotonic buffer.

    Science.gov (United States)

    Usaj, Marko; Kanduser, Masa

    2012-09-01

    The fusogenic state of the cell membrane can be induced by external electric field. When two fusogenic membranes are in close contact, cell fusion takes place. An appropriate hypotonic treatment of cells before the application of electric pulses significantly improves electrofusion efficiency. How hypotonic treatment improves electrofusion is still not known in detail. Our results indicate that at given induced transmembrane potential electroporation was not affected by buffer osmolarity. In contrast to electroporation, cells' response to hypotonic treatment significantly affects their electrofusion. High fusion yield was observed when B16-F1 cells were used; this cell line in hypotonic buffer resulted in 41 ± 9 % yield, while in isotonic buffer 32 ± 11 % yield was observed. Based on our knowledge, these fusion yields determined in situ by dual-color fluorescence microscopy are among the highest in electrofusion research field. The use of hypotonic buffer was more crucial for electrofusion of CHO cells; the fusion yield increased from below 1 % in isotonic buffer to 10 ± 4 % in hypotonic buffer. Since the same degree of cell permeabilization was achieved in both buffers, these results indicate that hypotonic treatment significantly improves fusion yield. The effect could be attributed to improved physical contact of cell membranes or to enhanced fusogenic state of the cell membrane itself.

  3. N- and C-terminal degradation of ecdysteroid receptor isoforms, when transiently expressed in mammalian CHO cells, is regulated by the proteasome and cysteine and threonine proteases.

    Science.gov (United States)

    Schauer, S; Burster, T; Spindler-Barth, M

    2012-06-01

    Transcriptional activity of nuclear receptors is the result of transactivation capability and the concentration of the receptor protein. The concentration of ecdysteroid receptor (EcR) isoforms, constitutively expressed in mammalian CHO cells, is dependent on a number of factors. As shown previously, ligand binding stabilizes receptor protein concentration. In this paper, we investigate the degradation of EcR isoforms and provide evidence that N-terminal degradation is modulated by isoform-specific ubiquitination sites present in the A/B domains of EcR-A and -B1. This was demonstrated by the increase in EcR concentration by treatment with carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), an inhibitor of ubiquitin-mediated proteasomal degradation and by deletion of ubiquitination sites. In addition, EcR is degraded by the peptidyl-dipeptidase cathepsin B (CatB) and the endopeptidase cathepsin S (CatS) at the C-terminus in an isoform-specific manner, despite identical C-termini. Ubiquitin-proteasome-mediated degradation and the proteolytic action are modulated by heterodimerization with Ultraspiracle (USP). The complex regulation of receptor protein concentration offers an additional opportunity to regulate transcriptional activity in an isoform- and target cell-specific way and allows the temporal limitation of hormone action. © 2012 The Authors. Insect Molecular Biology © 2012 The Royal Entomological Society.

  4. Changes in Al and Fe associated with amorphous soil minerals over one year after a wildfire at Pico Cho Marcial (Tenerife, Canary Islands, Spain

    Directory of Open Access Journals (Sweden)

    J. Notario

    2013-05-01

    Full Text Available Amorphous-linked Al, Fe and Si were determined both in burned and unburnt soil samples after a wildfire close to Pico Cho Marcial (Tenerife, Canary Islands, Spain that affected 7.1 ha of high mountain Teide broom scrub along four successive samplings held in September 2003 (three months after the wildfire, February 2004, June 2004 and October 2004. Soils in the area are Lithic Xerorthents, with a scarcely developed AC-type profile. The chemical elements under study were extracted using selective dissolutions (0.2M acid ammonium oxalate and 0.1N sodium pyrophosphate, and determined by Atomic Absorption Spectrophotometry. The average contents of total amorphous Al (oxalate-extractable and organo-metallic complexes-bound Al (pyrophosphate extractable were significantly higher in burned samples along the study. Also, the oxalate-extractable Al varied significantly along the different samplings, and so consequently did the Alp:Alox ratio. A progressive decrease in the Al:Si ratio in allophanes was also found throughout the study period. No differences were found for oxalate-extractable Fe, either between sample groups or samplings, which was also applicable to the (Alox+0.5Feox percentage.

  5. Development, qualification, validation and application of the neutral red uptake assay in Chinese Hamster Ovary (CHO) cells using a VITROCELL® VC10® smoke exposure system.

    Science.gov (United States)

    Fields, Wanda; Fowler, Kathy; Hargreaves, Victoria; Reeve, Lesley; Bombick, Betsy

    2017-04-01

    Cytotoxicity assessment of combustible tobacco products by neutral red uptake (NRU) has historically used total particulate matter (TPM) or solvent captured gas vapor phase (GVP), rather than fresh whole smoke. Here, the development, validation and application of the NRU assay in Chinese Hamster Ovary (CHO) cells, following exposure to fresh whole smoke generated with the VITROCELL® VC10® system is described. Whole smoke exposure is particularly important as both particulate and vapor phases of tobacco smoke show cytotoxicity in vitro. The VITROCELL® VC10® system provides exposure at the air liquid interface (ALI) to mimic in vivo conditions for assessing the toxicological impact of smoke in vitro. Instrument and assay validations are crucial for comparative analyses. 1) demonstrate functionality of the VITROCELL® VC10® system by installation, operational and performance qualification, 2) develop and validate a cellular system for assessing cytotoxicity following whole smoke exposure and 3) assess the whole smoke NRU assay sensitivity for statistical differentiation between a reference combustible cigarette (3R4F) and a primarily "heat-not-burn" cigarette (Eclipse). The VITROCELL® VC10® provided consistent generation and delivery of whole smoke; exposure-related changes in in vitro cytotoxicity were observed with reproducible IC50 values; comparative analysis showed that the heat-not-burn cigarette was significantly (P<0.001) less cytotoxic than the 3R4F combustible cigarette, consistent with the lower levels of chemical constituents liberated by primarily-heating the cigarette versus burning. Copyright © 2017. Published by Elsevier Ltd.

  6. Photolysis of CH{sub 3}CHO at 248 nm: Evidence of triple fragmentation from primary quantum yield of CH{sub 3} and HCO radicals and H atoms

    Energy Technology Data Exchange (ETDEWEB)

    Morajkar, Pranay; Schoemaecker, Coralie; Fittschen, Christa, E-mail: christa.fittschen@univ-lille1.fr [Université Lille Nord de France, PhysicoChimie des Processus de Combustion et de l’Atmosphère – PC2A, UMR 8522, F-59650 Villeneuve d’Ascq (France); Bossolasco, Adriana [Université Lille Nord de France, PhysicoChimie des Processus de Combustion et de l’Atmosphère – PC2A, UMR 8522, F-59650 Villeneuve d’Ascq (France); INFIQC (CONICET), Departamento de Fisicoquímica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba (Argentina)

    2014-06-07

    Radical quantum yields have been measured following the 248 nm photolysis of acetaldehyde, CH{sub 3}CHO. HCO radical and H atom yields have been quantified by time resolved continuous wave Cavity Ring Down Spectroscopy in the near infrared following their conversion to HO{sub 2} radicals by reaction with O{sub 2}. The CH{sub 3} radical yield has been determined using the same technique following their conversion into CH{sub 3}O{sub 2}. Absolute yields have been deduced for HCO radicals and H atoms through fitting of time resolved HO{sub 2} profiles, obtained under various O{sub 2} concentrations, to a complex model, while the CH{sub 3} yield has been determined relative to the CH{sub 3} yield from 248 nm photolysis of CH{sub 3}I. Time resolved HO{sub 2} profiles under very low O{sub 2} concentrations suggest that another unknown HO{sub 2} forming reaction path exists in this reaction system besides the conversion of HCO radicals and H atoms by reaction with O{sub 2}. HO{sub 2} profiles can be well reproduced under a large range of experimental conditions with the following quantum yields: CH{sub 3}CHO + hν{sub 248nm} → CH{sub 3}CHO{sup *}, CH{sub 3}CHO{sup *} → CH{sub 3} + HCO ϕ{sub 1a} = 0.125 ± 0.03, CH{sub 3}CHO{sup *} → CH{sub 3} + H + CO ϕ{sub 1e} = 0.205 ± 0.04, CH{sub 3}CHO{sup *}→{sup o{sub 2}}CH{sub 3}CO + HO{sub 2} ϕ{sub 1f} = 0.07 ± 0.01. The CH{sub 3}O{sub 2} quantum yield has been determined in separate experiments as ϕ{sub CH{sub 3}} = 0.33 ± 0.03 and is in excellent agreement with the CH{sub 3} yields derived from the HO{sub 2} measurements considering that the triple fragmentation (R1e) is an important reaction path in the 248 nm photolysis of CH{sub 3}CHO. From arithmetic considerations taking into account the HO{sub 2} and CH{sub 3} measurements we deduce a remaining quantum yield for the molecular pathway: CH{sub 3}CHO{sup *} → CH{sub 4} + CO ϕ{sub 1b} = 0.6. All experiments can be

  7. Vectorization, parallelization and porting of nuclear codes. Vectorization and parallelization. Progress report fiscal 1999

    Energy Technology Data Exchange (ETDEWEB)

    Adachi, Masaaki; Ogasawara, Shinobu; Kume, Etsuo [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment; Ishizuki, Shigeru; Nemoto, Toshiyuki; Kawasaki, Nobuo; Kawai, Wataru [Fujitsu Ltd., Tokyo (Japan); Yatake, Yo-ichi [Hitachi Ltd., Tokyo (Japan)

    2001-02-01

    Several computer codes in the nuclear field have been vectorized, parallelized and trans-ported on the FUJITSU VPP500 system, the AP3000 system, the SX-4 system and the Paragon system at Center for Promotion of Computational Science and Engineering in Japan Atomic Energy Research Institute. We dealt with 18 codes in fiscal 1999. These results are reported in 3 parts, i.e., the vectorization and the parallelization part on vector processors, the parallelization part on scalar processors and the porting part. In this report, we describe the vectorization and parallelization on vector processors. In this vectorization and parallelization on vector processors part, the vectorization of Relativistic Molecular Orbital Calculation code RSCAT, a microscopic transport code for high energy nuclear collisions code JAM, three-dimensional non-steady thermal-fluid analysis code STREAM, Relativistic Density Functional Theory code RDFT and High Speed Three-Dimensional Nodal Diffusion code MOSRA-Light on the VPP500 system and the SX-4 system are described. (author)

  8. Improved Coinfection with Amphotropic Pseudotyped Retroviral Vectors

    Directory of Open Access Journals (Sweden)

    Yuehong Wu

    2009-01-01

    Full Text Available Amphotropic pseudotyped retroviral vectors have typically been used to infect target cells without prior concentration. Although this can yield high rates of infection, higher rates may be needed where highly efficient coinfection of two or more vectors is needed. In this investigation we used amphotropic retroviral vectors produced by the Plat-A cell line and studied coinfection rates using green and red fluorescent proteins (EGFP and dsRed2. Target cells were primary human fibroblasts (PHF and 3T3 cells. Unconcentrated vector preparations produced a coinfection rate of ∼4% (defined as cells that are both red and green as a percentage of all cells infected. Optimized spinoculation, comprising centrifugation at 1200 g for 2 hours at 15∘C, increased the coinfection rate to ∼10%. Concentration by centrifugation at 10,000 g or by flocculation using Polybrene increased the coinfection rate to ∼25%. Combining the two processes, concentration by Polybrene flocculation and optimized spinoculation, increased the coinfection rate to 35% (3T3 or >50% (PHF. Improved coinfection should be valuable in protocols that require high transduction by combinations of two or more retroviral vectors.

  9. Could dark energy be vector-like?

    Science.gov (United States)

    Armendáriz-Picón, C.

    2004-07-01

    In this paper I explore whether a vector field can be the origin of the present stage of cosmic acceleration. In order to avoid violations of isotropy, the vector has be part of a 'cosmic triad', that is, a set of three identical vectors pointing in mutually orthogonal spatial directions. A triad is indeed able to drive a stage of late accelerated expansion in the universe, and there exist tracking attractors that render cosmic evolution insensitive to initial conditions. However, as in most other models, the onset of cosmic acceleration is determined by a parameter that has to be tuned to reproduce current observations. The triad equation of state can be sufficiently close to minus one today, and for tachyonic models it might be even less than that. I briefly analyse linear cosmological perturbation theory in the presence of a triad. It turns out that the existence of non-vanishing spatial vectors invalidates the decomposition theorem, i.e. scalar, vector and tensor perturbations do not decouple from each other. In a simplified case it is possible to analytically study the stability of the triad along the different cosmological attractors. The triad is classically stable during inflation, radiation and matter domination, but it is unstable during (late time) cosmic acceleration. I argue that this instability is not likely to have a significant impact at present.

  10. Evaluating the toxicity of bDtBPP on CHO-K1 cells for testing of single-use bioprocessing systems considering media selection, cell culture volume, mixing, and exposure duration.

    Science.gov (United States)

    Shah, Rhythm R; Linville, Taylor W; Whynot, Andrew D; Brazel, Christopher S

    2016-09-01

    Single-use bioprocessing bags are gaining popularity due to ease of use, lower risk of contamination, and ease of process scale-up. Bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP), a degradant of tris(2,4-di-tert-butylphenyl)phosphite, marketed as Irgafos 168®, which is an antioxidant stabilizer added to resins, has been identified as a potentially toxic leachate which may impact the performance of single-use, multilayer bioprocessing bags. In this study, the toxicity of bDtBPP was tested on CHO-K1 cells grown as adherent or suspended cells. The EC50 (effective concentration to cause 50% cell death) for adherent cells was found to be one order of magnitude higher than that for suspended CHO-K1 cells. While CHO-K1 cells had good cell viability when exposed to moderate concentrations of bDtBPP, the degradant was shown to impact the viable cell density (VCD) at much lower concentrations. Hence, in developing an industry-standard assay for testing the cytotoxicity of leachates, suspended cells (as commonly used in the bioprocessing industry) would likely be most sensitive, particularly when reporting EC50 values based on VCD. The effects of mixing, cell culture volume, and exposure duration were also evaluated for suspended CHO-K1 cells. It was found that the sensitivity of cell culture to leachates from single-use plastic bags was enhanced for suspended cells cultured for longer exposure times and when the cells were subjected to continuous agitation, both of which are important considerations in the production of biopharmaceuticals. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1318-1323, 2016. © 2016 American Institute of Chemical Engineers.

  11. Resultados de la técnica de Cho-Choo-Chop and Flip en la cirugía de catarata por facoemulsificación Results of the Cho-Choo-Chop and Flip's technique in the cataract surgery by phakoemulsification

    Directory of Open Access Journals (Sweden)

    Juan R Hernández Silva

    2005-06-01

    Full Text Available Se realizó un estudio descriptivo, prospectivo de corte transversal, cuyo universo estuvo constituido por todos los pacientes (ojos con diagnóstico de catarata presenil y senil que recibieron tratamiento quirúrgico con la técnica Cho-Choo-Chop and Flip por facoemulsificación en el Centro de Microcirugía Ocular en el período comprendido desde enero de 2003 hasta enero de 2005. Se seleccionó una muestra mediante un muestreo simple aleatorio de 198 pacientes donde la mayoría de los estudiados presentaban más de 65 años de edad. La agudeza visual con corrección alcanzada mejoró como promedio en 6 líneas en la cartilla de Snellen, con un astigmatismo inducido promedio de 0.43 D y el tiempo promedio de ultrasonido utilizado fue de 1,26 min, proporcional a la dureza del núcleo. Se presentaron un bajo número de complicaciones y fueron las más frecuentes la ruptura de cápsula posterior y la salida de vítreoA descriptive, prospective, cross-sectional study was conducted with all the patients (eyes with diagnosis of senile or presenile cataract that received surgical treatment with Cho-Choo-Chop and Flip's technique by phakoemulsification at the Center of Ocular Microsurgery from Janunary 2003 to January 2005. 198 patients were selected by randomized simple sampling. Most of the patietns studied were over 65 years old. The visual acuity with correction attained improved as an average in 6 lines according to Snellen's test, with an average induced astigmatism of 0.43 D. The average ultrasound time used was 1.26 min, proportional to the hardness of the nucleus. A few complications were observed. The rupture of the posterior capsule and the vitreous projection were the most frequent

  12. Emergence and Prevalence of Human Vector-Borne Diseases in Sink Vector Populations

    Science.gov (United States)

    Rascalou, Guilhem; Pontier, Dominique; Menu, Frédéric; Gourbière, Sébastien

    2012-01-01

    Vector-borne diseases represent a major public health concern in most tropical and subtropical areas, and an emerging threat for more developed countries. Our understanding of the ecology, evolution and control of these diseases relies predominantly on theory and data on pathogen transmission in large self-sustaining ‘source’ populations of vectors representative of highly endemic areas. However, there are numerous places where environmental conditions are less favourable to vector populations, but where immigration allows them to persist. We built an epidemiological model to investigate the dynamics of six major human vector borne-diseases in such non self-sustaining ‘sink’ vector populations. The model was parameterized through a review of the literature, and we performed extensive sensitivity analysis to look at the emergence and prevalence of the pathogen that could be encountered in these populations. Despite the low vector abundance in typical sink populations, all six human diseases were able to spread in 15–55% of cases after accidental introduction. The rate of spread was much more strongly influenced by vector longevity, immigration and feeding rates, than by transmission and virulence of the pathogen. Prevalence in humans remained lower than 5% for dengue, leishmaniasis and Japanese encephalitis, but substantially higher for diseases with longer duration of infection; malaria and the American and African trypanosomiasis. Vector-related parameters were again the key factors, although their influence was lower than on pathogen emergence. Our results emphasize the need for ecology and evolution to be thought in the context of metapopulations made of a mosaic of sink and source habitats, and to design vector control program not only targeting areas of high vector density, but working at a larger spatial scale. PMID:22629337

  13. Reducing the risk of adeno-associated virus (AAV) vector mobilization with AAV type 5 vectors.

    Science.gov (United States)

    Hewitt, F Curtis; Li, Chengwen; Gray, Steven J; Cockrell, Shelley; Washburn, Michael; Samulski, R Jude

    2009-04-01

    Current adeno-associated virus (AAV) gene therapy vectors package a transgene flanked by the terminal repeats (TRs) of AAV type 2 (AAV2). Although these vectors are replication deficient, wild-type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a type 2 TR (TR2)-flanked transgene in trans during superinfection by a helper virus, leading to "mobilization" of the vector genome from treated cells. More importantly, it appears likely that the majority of currently characterized AAV serotypes as well as the majority of new novel isolates are capable of rescuing and replicating AAV2 vector templates. To investigate this possibility, we flanked a green fluorescent protein transgene with type 2 and, the most divergent AAV serotype, type 5 TRs (TR2 or TR5). Consistent with AAV clades, AAV5 specifically replicated TR5 vectors, while AAV2 and AAV6 replicated TR2-flanked vectors. To exploit this specificity, we created a TR5 vector production system for Cap1 to Cap5. Next, we showed that persisting recombinant AAV genomes flanked by TR2s or TR5s were mobilized in vitro after addition of the cognate AAV Rep (as well as Rep6 for TR2) and adenoviral helper. Finally, we showed that a cell line containing a stably integrated wt AAV2 genome resulted in mobilization of a TR2-flanked vector but not a TR5-flanked vector upon adenoviral superinfection. Based on these data and the relative prevalence of wt AAV serotypes in the population, we propose that TR5 vectors have a significantly lower risk of mobilization and should be considered for clinical use.

  14. Emergence and prevalence of human vector-borne diseases in sink vector populations.

    Science.gov (United States)

    Rascalou, Guilhem; Pontier, Dominique; Menu, Frédéric; Gourbière, Sébastien

    2012-01-01

    Vector-borne diseases represent a major public health concern in most tropical and subtropical areas, and an emerging threat for more developed countries. Our understanding of the ecology, evolution and control of these diseases relies predominantly on theory and data on pathogen transmission in large self-sustaining 'source' populations of vectors representative of highly endemic areas. However, there are numerous places where environmental conditions are less favourable to vector populations, but where immigration allows them to persist. We built an epidemiological model to investigate the dynamics of six major human vector borne-diseases in such non self-sustaining 'sink' vector populations. The model was parameterized through a review of the literature, and we performed extensive sensitivity analysis to look at the emergence and prevalence of the pathogen that could be encountered in these populations. Despite the low vector abundance in typical sink populations, all six human diseases were able to spread in 15-55% of cases after accidental introduction. The rate of spread was much more strongly influenced by vector longevity, immigration and feeding rates, than by transmission and virulence of the pathogen. Prevalence in humans remained lower than 5% for dengue, leishmaniasis and Japanese encephalitis, but substantially higher for diseases with longer duration of infection; malaria and the American and African trypanosomiasis. Vector-related parameters were again the key factors, although their influence was lower than on pathogen emergence. Our results emphasize the need for ecology and evolution to be thought in the context of metapopulations made of a mosaic of sink and source habitats, and to design vector control program not only targeting areas of high vector density, but working at a larger spatial scale.

  15. Emergence and prevalence of human vector-borne diseases in sink vector populations.

    Directory of Open Access Journals (Sweden)

    Guilhem Rascalou

    Full Text Available Vector-borne diseases represent a major public health concern in most tropical and subtropical areas, and an emerging threat for more developed countries. Our understanding of the ecology, evolution and control of these diseases relies predominantly on theory and data on pathogen transmission in large self-sustaining 'source' populations of vectors representative of highly endemic areas. However, there are numerous places where environmental conditions are less favourable to vector populations, but where immigration allows them to persist. We built an epidemiological model to investigate the dynamics of six major human vector borne-diseases in such non self-sustaining 'sink' vector populations. The model was parameterized through a review of the literature, and we performed extensive sensitivity analysis to look at the emergence and prevalence of the pathogen that could be encountered in these populations. Despite the low vector abundance in typical sink populations, all six human diseases were able to spread in 15-55% of cases after accidental introduction. The rate of spread was much more strongly influenced by vector longevity, immigration and feeding rates, than by transmission and virulence of the pathogen. Prevalence in humans remained lower than 5% for dengue, leishmaniasis and Japanese encephalitis, but substantially higher for diseases with longer duration of infection; malaria and the American and African trypanosomiasis. Vector-related parameters were again the key factors, although their influence was lower than on pathogen emergence. Our results emphasize the need for ecology and evolution to be thought in the context of metapopulations made of a mosaic of sink and source habitats, and to design vector control program not only targeting areas of high vector density, but working at a larger spatial scale.

  16. Resonance vector soliton of the Rayleigh wave.

    Science.gov (United States)

    Adamashvili, G T

    2016-02-01

    A theory of acoustic vector solitons of self-induced transparency of the Rayleigh wave is constructed. A thin resonance transition layer on an elastic surface is considered using a model of a two-dimensional gas of impurity paramagnetic atoms or quantum dots. Explicit analytical expressions for the profile and parameters of the Rayleigh vector soliton with two different oscillation frequencies is obtained, as well as simulations of this nonlinear surface acoustic wave with realistic parameters, which can be used in acoustic experiments. It is shown that the properties of a surface vector soliton of the Rayleigh wave depend on the parameters of the resonance layer, the elastic medium, and the transverse structure of the surface acoustic wave.

  17. Phase interaction of short vector solitons

    Energy Technology Data Exchange (ETDEWEB)

    Aseeva, N.V., E-mail: naseeva@hse.ru [Higher School of Economics, Faculty of Business Informatics and Applied Mathematics, State University, Bolshaya Pecherskaya 25/12, 603155 Nizhny Novgorod (Russian Federation); Gromov, E.M.; Tyutin, V.V. [Higher School of Economics, Faculty of Business Informatics and Applied Mathematics, State University, Bolshaya Pecherskaya 25/12, 603155 Nizhny Novgorod (Russian Federation)

    2012-01-16

    An interaction of vector solitons in the frame of coupled third-order nonlinear Schrödinger equations taking into account third-order linear dispersion, nonlinear dispersion, and cross-phase modulation terms is considered. Phase nature of the solitons' interaction is shown. In particular, dependence of solitons' trajectories on initial distance between solitons is shown. Conditions of reflection and propagation of solitons through each other are obtained. -- Highlights: ► Short vector soliton's interaction in the frame of CTNSE without SRS is studied. ► Analytical and numerical approaches are considered. ► Phase effects lead to short vector soliton's interaction character change.

  18. On-demand tailored vector beams.

    Science.gov (United States)

    Perez-Garcia, Benjamin; López-Mariscal, Carlos; Hernandez-Aranda, Raul I; Gutiérrez-Vega, Julio C

    2017-08-20

    We introduce an effective optical system to produce optical beams with arbitrary, inhomogeneous polarization states. Using our system, we are capable of generating vector beams with discretionarily chosen transverse complex fields in a straightforward way. We generate several different instances of well-known vector beams and the less common spirally polarized vector beams, as well as a full Poincaré beam. We visually show the continual transition between azimuthally and radially polarized beams via a collection of spirally polarized beams. We experimentally determine the polarization states of the generated beams and quantitatively assess the performance of our system. We find that the measured polarization distributions accurately coincide with the intended input polarization distributions.

  19. Risk based surveillance for vector borne diseases

    DEFF Research Database (Denmark)

    Bødker, Rene

    an increasing trend in transmission potential over the last 25 years. However the model suggested that the climate in the Baltic See Region has always permitted transmission of these diseases. The model therefore suggests that a presently unknown factor until recently prevented introduction and spread......Increased temperatures and changes in rainfall pattern are likely to facilitate the spread and establishment of new vector borne diseases in the Baltic See Region. There are a large number of potential vector borne threats to the area. Existing endemic vector borne diseases are likely to increase...... and new exotic diseases like Usutu and West Nile Virus may lead to outbreaks in the region. In the worst case the combined effect of climate change and globalization may potentially lead to European outbreaks of important zoonotic mosquito borne infections like Rift Valley Fever in cattle and Japanese...

  20. Generating Series for Nested Bethe Vectors

    Directory of Open Access Journals (Sweden)

    Sergey Khoroshkin

    2008-11-01

    Full Text Available We reformulate nested relations between off-shell U_q(^gl_N Bethe vectors as a certain equation on generating series of strings of the composed U_q(^gl_N currents. Using inversion of the generating series we find a new type of hierarchical relations between universal off-shell Bethe vectors, useful for a derivation of Bethe equation. As an example of application, we use these relations for a derivation of analytical Bethe ansatz equations [Arnaudon D. et al., Ann. Henri Poincaré 7 (2006, 1217-1268, math-ph/0512037] for the parameters of universal Bethe vectors of the algebra U_q(^gl_2.

  1. Interactions between parasites and insects vectors

    Directory of Open Access Journals (Sweden)

    Hilary Hurd

    1994-01-01

    Full Text Available This review stresses the importance of studies that will provide a basic understanding of the pathology of parasite-infected vector insects. This knowledge should be a vital component of the very focussed initiatives currently being funded in the areas of vector control. Vector fecundity reduction is discussed as an example of such pathology. Underlying mechanisms are being investigated in a model system, Hymenolepis diminuta-infected Tenebrio molitor and in Onchocerca-infected blackflies and Plasmodium-infected Anopheles stephensi. In all cases, host vitellogenesis is disrupted by the parasite and, in the tapeworm/beetle model, interaction between the parasite and the endocrine control of the insect's reproductive physiology has been demonstrated.

  2. Efficient intracellular assembly of papillomaviral vectors.

    Science.gov (United States)

    Buck, Christopher B; Pastrana, Diana V; Lowy, Douglas R; Schiller, John T

    2004-01-01

    Although the papillomavirus structural proteins, L1 and L2, can spontaneously coassemble to form virus-like particles, currently available methods for production of L1/L2 particles capable of transducing reporter plasmids into mammalian cells are technically demanding and relatively low-yield. In this report, we describe a simple 293 cell transfection method for efficient intracellular production of papillomaviral-based gene transfer vectors carrying reporter plasmids. Using bovine papillomavirus type 1 (BPV1) and human papillomavirus type 16 as model papillomaviruses, we have developed a system for producing papillomaviral vector stocks with titers of several billion transducing units per milliliter. Production of these vectors requires both L1 and L2, and transduction can be prevented by papillomavirus-neutralizing antibodies. The stocks can be purified by an iodixanol (OptiPrep) gradient centrifugation procedure that is substantially more effective than standard cesium chloride gradient purification. Although earlier data had suggested a potential role for the viral early protein E2, we found that E2 protein expression did not enhance the intracellular production of BPV1 vectors. It was also possible to encapsidate reporter plasmids devoid of BPV1 DNA sequences. BPV1 vector production efficiency was significantly influenced by the size of the target plasmid being packaged. Use of 6-kb target plasmids resulted in BPV1 vector yields that were higher than those with target plasmids closer to the native 7.9-kb size of papillomavirus genomes. The results suggest that the intracellular assembly of papillomavirus structural proteins around heterologous reporter plasmids is surprisingly promiscuous and may be driven primarily by a size discrimination mechanism.

  3. Black hole superradiance signatures of ultralight vectors

    Science.gov (United States)

    Baryakhtar, Masha; Lasenby, Robert; Teo, Mae

    2017-08-01

    The process of superradiance can extract angular momentum and energy from astrophysical black holes (BHs) to populate gravitationally bound states with an exponentially large number of light bosons. We analytically calculate superradiant growth rates for vectors around rotating BHs in the regime where the vector Compton wavelength is much larger than the BH size. Spin-1 bound states have superradiance times as short as a second around stellar BHs, growing up to a thousand times faster than their spin-0 counterparts. The fast rates allow us to use measurements of rapidly spinning BHs in x-ray binaries to exclude a wide range of masses for weakly coupled spin-1 particles, 5 ×10-14-2 ×10-11 eV ; lighter masses in the range 6 ×10-20-2 ×10-17 eV start to be constrained by supermassive BH spin measurements at a lower level of confidence. We also explore routes to detection of new vector particles possible with the advent of gravitational wave (GW) astronomy. The LIGO-Virgo Collaboration could discover hints of a new light vector particle in statistical analyses of masses and spins of merging BHs. Vector annihilations source continuous monochromatic gravitational radiation which could be observed by current GW observatories. At design sensitivity, Advanced LIGO may measure up to thousands of annihilation signals from within the Milky Way, while hundreds of BHs born in binary mergers across the observable Universe may superradiate vector bound states and become new beacons of monochromatic gravitational waves.

  4. Application of Bred Vectors To Data Assimilation

    Science.gov (United States)

    Corazza, M.; Kalnay, E.; Patil, Dj

    We introduced a statistic, the BV-dimension, to measure the effective local finite-time dimensionality of the atmosphere. We show that this dimension is often quite low, and suggest that this finding has important implications for data assimilation and the accuracy of weather forecasting (Patil et al, 2001). The original database for this study was the forecasts of the NCEP global ensemble forecasting system. The initial differences between the control forecast and the per- turbed forecasts are called bred vectors. The control and perturbed initial conditions valid at time t=n(t are evolved using the forecast model until time t=(n+1) (t. The differences between the perturbed and the control forecasts are scaled down to their initial amplitude, and constitute the bred vectors valid at (n+1) (t. Their growth rate is typically about 1.5/day. The bred vectors are similar by construction to leading Lya- punov vectors except that they have small but finite amplitude, and they are valid at finite times. The original NCEP ensemble data set has 5 independent bred vectors. We define a local bred vector at each grid point by choosing the 5 by 5 grid points centered at the grid point (a region of about 1100km by 1100km), and using the north-south and east- west velocity components at 500mb pressure level to form a 50 dimensional column vector. Since we have k=5 global bred vectors, we also have k local bred vectors at each grid point. We estimate the effective dimensionality of the subspace spanned by the local bred vectors by performing a singular value decomposition (EOF analysis). The k local bred vector columns form a 50xk matrix M. The singular values s(i) of M measure the extent to which the k column unit vectors making up the matrix M point in the direction of v(i). We define the bred vector dimension as BVDIM={Sum[s(i)]}^2/{Sum[s(i)]^2} For example, if 4 out of the 5 vectors lie along v, and one lies along v, the BV- dimension would be BVDIM[sqrt(4), 1, 0

  5. Integrated vector management for malaria control

    Directory of Open Access Journals (Sweden)

    Impoinvil Daniel E

    2008-12-01

    Full Text Available Abstract Integrated vector management (IVM is defined as "a rational decision-making process for the optimal use of resources for vector control" and includes five key elements: 1 evidence-based decision-making, 2 integrated approaches 3, collaboration within the health sector and with other sectors, 4 advocacy, social mobilization, and legislation, and 5 capacity-building. In 2004, the WHO adopted IVM globally for the control of all vector-borne diseases. Important recent progress has been made in developing and promoting IVM for national malaria control programmes in Africa at a time when successful malaria control programmes are scaling-up with insecticide-treated nets (ITN and/or indoor residual spraying (IRS coverage. While interventions using only ITNs and/or IRS successfully reduce transmission intensity and the burden of malaria in many situations, it is not clear if these interventions alone will achieve those critical low levels that result in malaria elimination. Despite the successful employment of comprehensive integrated malaria control programmes, further strengthening of vector control components through IVM is relevant, especially during the "end-game" where control is successful and further efforts are required to go from low transmission situations to sustained local and country-wide malaria elimination. To meet this need and to ensure sustainability of control efforts, malaria control programmes should strengthen their capacity to use data for decision-making with respect to evaluation of current vector control programmes, employment of additional vector control tools in conjunction with ITN/IRS tactics, case-detection and treatment strategies, and determine how much and what types of vector control and interdisciplinary input are required to achieve malaria elimination. Similarly, on a global scale, there is a need for continued research to identify and evaluate new tools for vector control that can be integrated with

  6. Vector domain decomposition schemes for parabolic equations

    Science.gov (United States)

    Vabishchevich, P. N.

    2017-09-01

    A new class of domain decomposition schemes for finding approximate solutions of timedependent problems for partial differential equations is proposed and studied. A boundary value problem for a second-order parabolic equation is used as a model problem. The general approach to the construction of domain decomposition schemes is based on partition of unity. Specifically, a vector problem is set up for solving problems in individual subdomains. Stability conditions for vector regionally additive schemes of first- and second-order accuracy are obtained.

  7. [Conflicts and vector-borne diseases].

    Science.gov (United States)

    Bygbjerg, Ib Christian

    2010-01-11

    Based on literature and personal experiences, vector-borne diseases and conflicts are reviewed. Simple rapid diagnostic tests for three important parasitoses are available. Resort is often made to case definitions and to presumptive treatment. Resistance is an emerging problem. Vaccines are still not available for most diseases. Promising preventive methods, including long-lasting impregnated bed-nets and tents, are available. War has been an impetus for disclosing life-cycles of vector-borne diseases and for control methods; peace, reconciliation and poverty reduction are required to achieve lasting control.

  8. Vectorized algorithms for spiking neural network simulation.

    Science.gov (United States)

    Brette, Romain; Goodman, Dan F M

    2011-06-01

    High-level languages (Matlab, Python) are popular in neuroscience because they are flexible and accelerate development. However, for simulating spiking neural networks, the cost of interpretation is a bottleneck. We describe a set of algorithms to simulate large spiking neural networks efficiently with high-level languages using vector-based operations. These algorithms constitute the core of Brian, a spiking neural network simulator written in the Python language. Vectorized simulation makes it possible to combine the flexibility of high-level languages with the computational efficiency usually associated with compiled languages.

  9. Retrotransposon vectors for gene delivery in plants

    Directory of Open Access Journals (Sweden)

    Hou Yi

    2010-08-01

    Full Text Available Abstract Background Retrotransposons are abundant components of plant genomes, and although some plant retrotransposons have been used as insertional mutagens, these mobile genetic elements have not been widely exploited for plant genome manipulation. In vertebrates and yeast, retrotransposons and retroviruses are routinely altered to carry additional genes that are copied into complementary (cDNA through reverse transcription. Integration of cDNA results in gene delivery; recombination of cDNA with homologous chromosomal sequences can create targeted gene modifications. Plant retrotransposon-based vectors, therefore, may provide new opportunities for plant genome engineering. Results A retrotransposon vector system was developed for gene delivery in plants based on the Tnt1 element from Nicotiana tabacum. Mini-Tnt1 transfer vectors were constructed that lack coding sequences yet retain the 5' and 3' long terminal repeats (LTRs and adjacent cis sequences required for reverse transcription. The internal coding region of Tnt1 was replaced with a neomycin phosphotransferase gene to monitor replication by reverse transcription. Two different mini-Tnt1 s were developed: one with the native 5' LTR and the other with a chimeric 5' LTR that had the first 233 bp replaced by the CaMV 35 S promoter. After transfer into tobacco protoplasts, both vectors undergo retrotransposition using GAG and POL proteins provided in trans by endogenous Tnt1 elements. The transposition frequencies of mini-Tnt1 vectors are comparable with native Tnt1 elements, and like the native elements, insertion sites are within or near coding sequences. In this paper, we provide evidence that template switching occurs during mini-Tnt1 reverse transcription, indicating that multiple copies of Tnt1 mRNA are packaged into virus-like particles. Conclusions Our data demonstrate that mini-Tnt1 vectors can replicate efficiently in tobacco cells using GAG and POL proteins provided in trans by

  10. Variable ordering structures in vector optimization

    CERN Document Server

    Eichfelder, Gabriele

    2014-01-01

    This book provides an introduction to vector optimization with variable ordering structures, i.e., to optimization problems with a vector-valued objective function where the elements in the objective space are compared based on a variable ordering structure: instead of a partial ordering defined by a convex cone, we see a whole family of convex cones, one attached to each element of the objective space. The book starts by presenting several applications that have recently sparked new interest in these optimization problems, and goes on to discuss fundamentals and important results on a wide ra

  11. Vector Fields European user group meeting

    CERN Multimedia

    2007-01-01

    The "Vector Fields European user group meeting" will take place at CERN on 26 and 27 September 2007. Within this framework two workshops are organized at the CERN Training Centre: 24 September 2007
 Modelling Magnets with Opera 25 September 2007
Modelling of Charged Particle Beam Devices with Opera If you are interested in attending the workshop or the user group meeting please contact Julie Shepherd (Vector Fields) or Pierre Baehler (CERN) directly at: Julie.Shepherd@vectorfields.co.uk, +44 (0) 1865 854933 or +44 (0) 1865 370151 Pierre.Baehler@cern.ch, 75016 / 160156.

  12. Learning and Memory in Disease Vector Insects.

    Science.gov (United States)

    Vinauger, Clément; Lahondère, Chloé; Cohuet, Anna; Lazzari, Claudio R; Riffell, Jeffrey A

    2016-10-01

    Learning and memory plays an important role in host preference and parasite transmission by disease vector insects. Historically there has been a dearth of standardized protocols that permit testing their learning abilities, thus limiting discussion on the potential epidemiological consequences of learning and memory to a largely speculative extent. However, with increasing evidence that individual experience and associative learning can affect processes such as oviposition site selection and host preference, it is timely to review the recently acquired knowledge, identify research gaps and discuss the implication of learning in disease vector insects in perspective with control strategies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Constraints on vector meson photoproduction spin observables

    Science.gov (United States)

    Kloet, W. M.; Tabakin, Frank

    2000-01-01

    Extraction of spin observables from vector meson photoproduction on a nucleon target is described. Starting from density matrix elements in the vector meson's rest frame, we transform to spin observables in the photon-nucleon c.m. frame. Several constraints on the transformed density matrix and on the spin observables follow from requiring that the angular distribution and the density matrix be positive definite. A set of constraints that are required in order to extract meaningful spin observables from forthcoming data are enunciated.

  14. Vector coherent states for nanoparticle systems

    Energy Technology Data Exchange (ETDEWEB)

    Aremua, Isiaka [Institut de Mathematiques et de Sciences Physiques (IMSP), University of Abomey-Calavi, 01 BP 613 Porto-Novo (Benin); Hounkonnou, Mahouton Norbert, E-mail: iaremua@imsp-uac.org, E-mail: norbert.hounkonnou@cipma.uac.bj [International Chair of Mathematical Physics and Applications (ICMPA-UNESCO Chair), University of Abomey-Calavi, 072 BP 50 Cotonou (Benin)

    2011-11-18

    The first part of this work deals with a formalism of vector coherent states construction for a system of M Fermi-type modes associated with N bosonic modes. Then follows a generalization to a Hamiltonian describing the translational motion of the center of mass of a nanoparticle. The latter gives rise to a new mechanism for the electronic energy relaxation in nanocrystals, intensively studied today in condensed matter physics. Finite degeneracies of the involved Hamiltonian systems are also investigated. The defined vector coherent states satisfy relevant mathematical properties of continuity, resolution of identity, temporal stability and action identity. (paper)

  15. Vector Fields and Flows on Differentiable Stacks

    DEFF Research Database (Denmark)

    A. Hepworth, Richard

    2009-01-01

    and uniqueness of flows on a manifold as well as the author's existing results for orbifolds. It sets the scene for a discussion of Morse Theory on a general proper stack and also paves the way for the categorification of other key aspects of differential geometry such as the tangent bundle and the Lie algebra......This paper introduces the notions of vector field and flow on a general differentiable stack. Our main theorem states that the flow of a vector field on a compact proper differentiable stack exists and is unique up to a uniquely determined 2-cell. This extends the usual result on the existence...

  16. The Cross Product of Two Vectors Is Not Just Another Vector--A Major Misconception Being Perpetuated in Calculus and Vector Analysis Textbooks.

    Science.gov (United States)

    Elk, Seymour B.

    1997-01-01

    Suggests that the cross product of two vectors can be more easily and accurately explained by starting from the perspective of dyadics because then the concept of vector multiplication has a simple geometrical picture that encompasses both the dot and cross products in any number of dimensions in terms of orthogonal unit vector components. (AIM)

  17. [Sendai virus vector: vector development and its application to health care and biotechnology].

    Science.gov (United States)

    Iida, Akihiro

    2007-06-01

    Sendai virus (SeV) is an enveloped virus with a nonsegmented negative-strand RNA genome and a member of the paramyxovirus family. We have developed SeV vector which has shown a high efficiently of gene transfer and expression of foreign genes to a wide range of dividing and non-dividing mammalian cells and tissues. One of the characteristics of the vector is that the genome is located exclusively in the cytoplasm of infected cells and does not go through a DNA phase; thus there is no concern about unwanted integration of foreign sequences into chromosomal DNA. Therefore, this new class of "cytoplasmic RNA vector", an RNA vector with cytoplasmic expression, is expected to be a safer and more efficient viral vector than existing vectors for application to human therapy in various fields including gene therapy and vaccination. In this review, I describe development of Sendai virus vector, its application in the field of biotechnology and clinical application aiming to treat for a large number of diseases including cancer, cardiovascular disease, infectious diseases and neurologic disorders.

  18. Host and vector-dependent effects on the risk of germline transmission of AAV vectors.

    Science.gov (United States)

    Favaro, Patricia; Downey, Harre D; Zhou, J Shangzhen; Wright, J Fraser; Hauck, Bernd; Mingozzi, Federico; High, Katherine A; Arruda, Valder R

    2009-06-01

    The assessment of the risk of germline transmission of vector-coded sequences is critical for clinical translation of gene transfer strategies. We used rabbit models to analyze the risk of germline transmission of adeno-associated viral (AAV) vectors. Intravenous injection of AAV-2 or AAV-8 resulted in liver-mediated, long-term expression of therapeutic levels of human factor IX (hFIX) in a dose-dependent manner. In high-dose cohorts, AAV-8 resulted in twofold higher levels of circulating hFIX and of vector DNA in liver compared to AAV-2. Vector sequences were found in the semen of all rabbits. The kinetics of vector clearance from semen was dose- and time-dependent but serotype-independent. No late recurrence of AAV-8 sequences was found in the semen over several consecutive cycles of spermatogenesis. In a novel rabbit model, AAV-2 or AAV-8 sequences were detected in the semen of vasectomized animals that lack germ cells. Therefore, structures of the genitourinary (GU) tract, as well as the testis, contribute significantly to vector shedding in the semen. Collectively, data from these two models suggest that the risk of inadvertent germline transmission in males by AAV-8 vectors is low, similar to that of AAV-2, and that AAV dissemination to the semen is in part modulated by host-dependent factors.

  19. Integrating Transgenic Vector Manipulation with Clinical Interventions to Manage Vector-Borne Diseases.

    Science.gov (United States)

    Okamoto, Kenichi W; Gould, Fred; Lloyd, Alun L

    2016-03-01

    Many vector-borne diseases lack effective vaccines and medications, and the limitations of traditional vector control have inspired novel approaches based on using genetic engineering to manipulate vector populations and thereby reduce transmission. Yet both the short- and long-term epidemiological effects of these transgenic strategies are highly uncertain. If neither vaccines, medications, nor transgenic strategies can by themselves suffice for managing vector-borne diseases, integrating these approaches becomes key. Here we develop a framework to evaluate how clinical interventions (i.e., vaccination and medication) can be integrated with transgenic vector manipulation strategies to prevent disease invasion and reduce disease incidence. We show that the ability of clinical interventions to accelerate disease suppression can depend on the nature of the transgenic manipulation deployed (e.g., whether vector population reduction or replacement is attempted). We find that making a specific, individual strategy highly effective may not be necessary for attaining public-health objectives, provided suitable combinations can be adopted. However, we show how combining only partially effective antimicrobial drugs or vaccination with transgenic vector manipulations that merely temporarily lower vector competence can amplify disease resurgence following transient suppression. Thus, transgenic vector manipulation that cannot be sustained can have adverse consequences-consequences which ineffective clinical interventions can at best only mitigate, and at worst temporarily exacerbate. This result, which arises from differences between the time scale on which the interventions affect disease dynamics and the time scale of host population dynamics, highlights the importance of accounting for the potential delay in the effects of deploying public health strategies on long-term disease incidence. We find that for systems at the disease-endemic equilibrium, even modest

  20. Broad patterns in domestic vector-borne Trypanosoma cruzi transmission dynamics: synanthropic animals and vector control.

    Science.gov (United States)

    Peterson, Jennifer K; Bartsch, Sarah M; Lee, Bruce Y; Dobson, Andrew P

    2015-10-22

    Chagas disease (caused by Trypanosoma cruzi) is the most important neglected tropical disease (NTD) in Latin America, infecting an estimated 5.7 million people in the 21 countries where it is endemic. It is one of the NTDs targeted for control and elimination by the 2020 London Declaration goals, with the first goal being to interrupt intra-domiciliary vector-borne T. cruzi transmission. A key question in domestic T. cruzi transmission is the role that synanthropic animals play in T. cruzi transmission to humans. Here, we ask, (1) do synanthropic animals need to be targeted in Chagas disease prevention policies?, and (2) how does the presence of animals affect the efficacy of vector control? We developed a simple mathematical model to simulate domestic vector-borne T. cruzi transmission and to specifically examine the interaction between the presence of synanthropic animals and effects of vector control. We used the model to explore how the interactions between triatomine bugs, humans and animals impact the number and proportion of T. cruzi-infected bugs and humans. We then examined how T. cruzi dynamics change when control measures targeting vector abundance are introduced into the system. We found that the presence of synanthropic animals slows the speed of T. cruzi transmission to humans, and increases the sensitivity of T. cruzi transmission dynamics to vector control measures at comparable triatomine carrying capacities. However, T. cruzi transmission is amplified when triatomine carrying capacity increases with the abundance of syntathoropic hosts. Our results suggest that in domestic T. cruzi transmission scenarios where no vector control measures are in place, a reduction in synanthropic animals may slow T. cruzi transmission to humans, but it would not completely eliminate transmission. To reach the 2020 goal of interrupting intra-domiciliary T. cruzi transmission, it is critical to target vector populations. Additionally, where vector control measures

  1. New Constraints on Light Vectors Coupled to Anomalous Currents

    Science.gov (United States)

    Dror, Jeff A.; Lasenby, Robert; Pospelov, Maxim

    2017-10-01

    We derive new constraints on light vectors coupled to standard model (SM) fermions, when the corresponding SM current is broken by the chiral anomaly. The cancellation of the anomaly by heavy fermions results, in the low-energy theory, in Wess-Zumino-type interactions between the new vector and the SM gauge bosons. These interactions are determined by the requirement that the heavy sector preserves the SM gauge groups and lead to (energy /vector mass )2 enhanced rates for processes involving the longitudinal mode of the new vector. Taking the example of a vector coupled to a vector coupled to SM baryon number, Z decays and flavor-changing neutral current meson decays via the new vector can occur with (weak scale /vector mass )2 enhanced rates. These processes place significantly stronger coupling bounds than others considered in the literature, over a wide range of vector masses.

  2. Effect of genome size on AAV vector packaging.

    Science.gov (United States)

    Wu, Zhijian; Yang, Hongyan; Colosi, Peter

    2010-01-01

    Adeno-associated virus (AAV) vector genomes have been limited to 5 kilobases (kb) in length because their packaging limit was thought to be similar to the size of the parent AAV genome. Recent reports claim that significantly larger vector genomes can be packaged intact. We examined the packaged vector genomes from plasmid-encoded AAV vectors that ranged from 4.7 to 8.7 kb in length, using AAV types 2, 5, and 8 capsids. Southern blot analysis indicated that packaged AAV vector genomes never exceeded 5.2 kb in length irrespective of the size of the plasmid-encoded vector or the capsid type. This result was confirmed by vector genome probing with strand-specific oligonucleotides. The packaged vector genomes derived from plasmid-encoded vectors exceeding 5 kb were heterogeneous in length and truncated on the 5' end. Despite their truncated genomes, vector preparations produced from plasmid-encoded vectors exceeding 5.2 kb mediated reporter gene expression in vitro at high multiplicity of infection (MOI). The efficiency of expression was substantially lower than that of reporter vectors with genomes <5 kb in length. We propose that transcriptionally functional, intact vector genomes are generated in cells transduced at high MOI from the fragmentary genomes of these larger vectors, probably by recombination.

  3. Method for transforming a feature vector

    NARCIS (Netherlands)

    Veldhuis, Raymond N.J.; Chen, C.; Kevenaar, Thomas A.M.; Akkermans, Antonius H.M.

    2007-01-01

    The present invention relates to a method for transforming a feature vector comprising a first and a second feature represented by a first and a second feature value, respectively, into a feature code using an encoder, said feature code usable in an algorithm and having a predetermined number of

  4. Learning Latent Vector Spaces for Product Search

    NARCIS (Netherlands)

    Van Gysel, C.; de Rijke, M.; Kanoulas, E.

    2016-01-01

    We introduce a novel latent vector space model that jointly learns the latent representations of words, e-commerce products and a mapping between the two without the need for explicit annotations. The power of the model lies in its ability to directly model the discriminative relation between

  5. Ecology of parasite-vector interactions

    NARCIS (Netherlands)

    Takken, W.; Koenraadt, C.J.M.

    2013-01-01

    Vector-borne diseases continue to be one of the most important determinants affecting human and animal health. Large numbers of people suffer from diseases like malaria, dengue, filariasis and leishmaniasis, especially in the tropics. Whereas these diseases were eradicated from the temperate climate

  6. Bethe vectors for XXX-spin chain

    Science.gov (United States)

    Burdík, Čestmír; Fuksa, Jan; Isaev, Alexei

    2014-11-01

    The paper deals with algebraic Bethe ansatz for XXX-spin chain. Generators of Yang-Baxter algebra are expressed in basis of free fermions and used to calculate explicit form of Bethe vectors. Their relation to N-component models is used to prove conjecture about their form in general. Some remarks on inhomogeneous XXX-spin chain are included.

  7. Angry Birds Mathematics: Parabolas and Vectors

    Science.gov (United States)

    Lamb, John H.

    2013-01-01

    John Lamb, a professor of mathematics education and a teacher of high school precalculus, describes how he developed a way to use the elements of the game Angry Birds® as a platform to engage his students with the concepts of parabolas and vectors. The game could be categorized as a type of microworld game in which students interact with the…

  8. Vector boson production in hadron nuclear collisions

    Energy Technology Data Exchange (ETDEWEB)

    Walker, W.D. (Duke Univ., Durham, NC (USA)); Whitmore, J. (Pennsylvania State Univ., University Park, PA (USA). Lab. for Elementary Particle Science); Toothacker, W.S. (Pennsylvania State Univ., Mont Alto (USA)); Hill, J.C.; Neale, W.W. (Cambridge Univ. (UK)); Lucas, P.; Voyvodic, L. (Fermi National Accelerator Lab., Batavia, IL (USA)); Ammar, R.; Gress, J. (Kansas Univ., Lawrence (USA)); Bishop, J.M.; Biswas, N.N.; Cason, N.M.; Mattingly, M.C.K.; Ruchti, R.C.; Shephard, W.D. (Notre Dame Univ., IN (USA))

    1991-01-31

    We report a search for the production of light quark vector bosons in hadron-nucleus collisions at 100 GeV bombarding energy. We find surprisingly few of these resonances produced. The lack of these particles is though to be due to the absorption by the many modestly energetic nucleons and the few anti-nucleons in the final state. (orig.).

  9. Malaria vector control: current and future strategies

    NARCIS (Netherlands)

    Takken, W.; Knols, B.G.J.

    2009-01-01

    The recently announced call for malaria eradication represents a new page in the history of this disease. This has been triggered by remarkable reductions in malaria resulting from combined application of effective drugs and vector control. However, this strategy is threatened by development of

  10. Generating and measuring nondiffracting vector Bessel beams

    CSIR Research Space (South Africa)

    Dudley, Angela L

    2013-09-01

    Full Text Available modulator and an azimuthally varying birefringent plate, known as a q-plate. We extend our control of both the geometric and dynamic phases to perform a polarization and modal decomposition on the vector field. We study both single-charged Bessel beams...

  11. NLO vector boson production with light jets

    CERN Document Server

    Bern, Z; Dixon, L J; Cordero, F Febres; Forde, D; Gleisberg, T; Hoeche, S; Ita, H; Kosower, D A; Maitre, D; Ozeren, K

    2012-01-01

    In this contribution we present recent progress in the computation of next-to-leading order (NLO) QCD corrections for the production of an electroweak vector boson in association with jets at hadron colliders. We focus on results obtained using the virtual matrix element library BLACKHAT in conjunction with SHERPA, focusing on results relevant to understanding the background to top production.

  12. Linearization of germs of hyperbolic vector fields

    NARCIS (Netherlands)

    Bonckaert, P; Naudot, [No Value; Yang, JZ

    2003-01-01

    We develop a normal form to express asymptotically a conjugacy between a germ of resonant vector field and its linear part. We show that such an asymptotic expression can be written in terms of functions of the Logarithmic Mourtada type. To cite this article: P Bonckaert et al., C. R. Acad. Sci.

  13. Perturbations of ultralight vector field dark matter

    CERN Document Server

    Cembranos, J A R; Jareño, S J Núñez

    2016-01-01

    We study the dynamics of cosmological perturbations in models of dark matter based on ultralight coherent vector fields. Very much as for scalar field dark matter, we find two different regimes in the evolution: for modes with $k^2\\ll {\\cal H}ma$, we have a particle-like behaviour indistinguishable from cold dark matter, whereas for modes with $k^2\\gg {\\cal H}ma$, we get a wave-like behaviour in which the sound speed is non-vanishing and of order $c_s^2\\simeq k^2/m^2a^2$. This implies that, also in these models, structure formation could be suppressed on small scales. However, unlike the scalar case, the fact that the background evolution contains a non-vanishing homogeneous vector field implies that, in general, the evolution of the three kinds of perturbations (scalar, vector and tensor) can no longer be decoupled at the linear level. More specifically, in the particle regime, the three types of perturbations are actually decoupled, whereas in the wave regime, the three vector field perturbations generate o...

  14. Efficient Multiplicative Updates for Support Vector Machines

    DEFF Research Database (Denmark)

    Potluru, Vamsi K.; Plis, Sergie N; Mørup, Morten

    2009-01-01

    The dual formulation of the support vector machine (SVM) objective function is an instance of a nonnegative quadratic programming problem. We reformulate the SVM objective function as a matrix factorization problem which establishes a connection with the regularized nonnegative matrix factorizati...

  15. Generalized Derivative Based Kernelized Learning Vector Quantization

    NARCIS (Netherlands)

    Schleif, Frank-Michael; Villmann, Thomas; Hammer, Barbara; Schneider, Petra; Biehl, Michael; Fyfe, Colin; Tino, Peter; Charles, Darryl; Garcia-Osoro, Cesar; Yin, Hujun

    2010-01-01

    We derive a novel derivative based version of kernelized Generalized Learning Vector Quantization (KGLVQ) as an effective, easy to interpret, prototype based and kernelized classifier. It is called D-KGLVQ and we provide generalization error bounds, experimental results on real world data, showing

  16. [Conflicts and vector-borne diseases

    DEFF Research Database (Denmark)

    Bygbjerg, Ib Christian

    2010-01-01

    not available for most diseases. Promising preventive methods, including long-lasting impregnated bed-nets and tents, are available. War has been an impetus for disclosing life-cycles of vector-borne diseases and for control methods; peace, reconciliation and poverty reduction are required to achieve lasting...

  17. Closed-chain principal vector linkages

    NARCIS (Netherlands)

    van der Wijk, V.; Flores, Paulo; Viadero, Fernando

    2015-01-01

    For high-speed robotics dynamic balance is an important property for low base vibrations and short cycle times. To consider dynamic balance in the beginning of the design process of a manipulator, mechanism solutions can be synthesized from principal vector linkages, which are fundamental kinematic

  18. Artificial neural networks and support vector mac

    Indian Academy of Sciences (India)

    Quantitative structure-property relationships of electroluminescent materials: Artificial neural networks and support vector machines to predict electroluminescence of organic molecules. ALANA FERNANDES GOLIN and RICARDO STEFANI. ∗. Laboratório de Estudos de Materiais (LEMAT), Instituto de Ciências Exatas e da ...

  19. Creating and decomposing vector Bessel beams

    CSIR Research Space (South Africa)

    Dudley, Angela L

    2013-07-01

    Full Text Available -1 58th Annual SAIP Conference, Richards Bay, 8-12 July 2013 Creating and decomposing vector Bessel beams Angela Dudley1,*, Yanming Li2, Thandeka Mhlanga1, Michael Escuti2 and Andrew Forbes1 1 CSIR National Laser Centre, Pretoria, South...

  20. High performance computing on vector systems

    CERN Document Server

    Roller, Sabine

    2008-01-01

    Presents the developments in high-performance computing and simulation on modern supercomputer architectures. This book covers trends in hardware and software development in general and specifically the vector-based systems and heterogeneous architectures. It presents innovative fields like coupled multi-physics or multi-scale simulations.